LIMD1, a tumour suppressor located at chromosome 3p21.3, is frequently lost in non-small-cell lung cancer, yet its role in tumour-immune interactions remains unclear. Here, we show LIMD1 loss increases PD-L1 protein abundance across multiple lung cancer models and primary airway epithelial cells. Mechanistically, LIMD1 restrains PD-L1 through post-transcriptional and post-translational mechanisms. LIMD1 loss can relieve microRNA-mediated repression of the CD274 3'UTR, and LIMD1 loss can also disrupt ARIH1-PD-L1 association, reduce PD-L1 polyubiquitination, and stabilise PD-L1 protein without a commensurate increase in CD274 transcript levels in isogenic models. Functionally, LIMD1-deficient tumour cells suppress CD8+ T-cell activation in vitro and show enhanced sensitivity to PD-1/PD-L1 blockade in tumour-PBMC co-culture assays. Analysis of TRACERx non-small-cell lung cancer samples revealed clonal LIMD1 loss of heterozygosity in ∼40% of lung adenocarcinomas, where it is associated with increased tumour PD-L1 expression. Across independent patient cohorts receiving immune checkpoint blockade, low LIMD1 expression was enriched among responders. We identify LIMD1 as a tumour-intrinsic regulator of PD-L1 turnover and suggest that tumour suppressor loss can shape immune checkpoint biology and influence immunotherapy response.

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. ### Competing Interest Statement B.R.S. is an inventor on patents and patent applications involving ferroptosis; co-founded and serves as a consultant to ProJenX, Inc. and Exarta Therapeutics; holds equity in Sonata Therapeutics; serves as a consultant to Weatherwax Biotechnologies Corporation and Akin Gump Strauss Hauer& Feld LLP. The remaining authors declare no competing interests. Barts Charity, G-002509 Cancer Research UK, C355/A25137, C7893/A26233, CTRQQR-2021\100004 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/V009567/1

Immune checkpoint inhibitors (ICIs) are extremely effective in a subgroup of mismatch repair-deficient (MMRd) cancers, but ∼50% remain resistant to treatment. We have shown for the first time that this may be due to the differential regulation of factors linked to response to ICIs upon loss of the different MMR genes. Here, we show that increased PD-L1 expression is observed upon loss of the MMR genes MLH1, MSH2 and PMS2. However, this is not true upon loss of MSH6, and we show that this is due to a novel role for MSH6 as a direct regulator of PD-L1 transcription, dependent on recruitment by the histone trimethyltransferase SETD2. Next-generation sequencing of MLH1 and MSH6 knockout (KO) cells revealed that MSH6 KO cells have significantly lower microsatellite instability in comparison to MLH1 KO cells, despite MSH6 KO cells having a higher mutational burden. These findings emphasise the need for gene-specific stratification in the MMRd cohort.

Hypoxia-inducible factors (HIFs) orchestrate cellular responses to oxygen deprivation and are frequently activated in triple-negative breast cancer (TNBC), driving metastasis and poor prognosis[1][1],[2][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 tissues[3][3],[4][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-1α and HIF-2α independently of the canonical oxygen sensing pathway. Proximity-labelling proteomics revealed that CoAsy deficiency disrupts the association between HIF-1α and the proteasome through UBFD1, a novel CoA-binding protein that scaffolds HIFα for degradation. UBFD1’s CoA-dependent interaction with HIF-α 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. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4

MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the N- and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous C-terminal tag fusions without requiring homology arms. Using this system, we generated the first C-terminal HaloTag fusion of AGO2 (AGO2HALO) in human A549 cells. We found that the AGO2HALO fusion protein exhibits reduced binding with TNRC6A, with no effect on cell viability. However, it significantly impairs RNA cleavage, silencing activity, and nuclear localization. We further compared AGO2-EGFP and EGFP-AGO2 using transient transfection. N-terminally tagged AGO2 retained wild-type-like function and localization, while C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing, nuclear import, and P-body localization. These results demonstrate that a C-terminal HaloTag compromises AGO2 functionality and is unsuitable for studying RISC biology. Our findings highlight the importance of validating tagging strategies to avoid misleading conclusions due to tag-induced functional defects. Pre-print, bioRxiv.

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. ![Figure][1] LIMD1 defines the scope of miRNA-mediated targeting and repression ### Competing Interest Statement K.S. and D.C. are employees of Eclipse BioInnovations. The other authors declare no competing interests. [1]: pending:yes

ABSTRACTSTAG proteins are key regulators of the cohesin complex and are often linked to alterations in cell identity and disease. Among the mammalian STAG paralogs, STAG3 has been less extensively studied beyond its known roles in meiosis. In this work, we demonstrate that STAG3 is expressed in mouse embryonic stem cells (mESCs) and primordial germ cell-like cells (PGCLCs), where it is required for cell fate decisions. Distinct from the other STAG proteins, STAG3 mediates its effects in the cytoplasm, facilitating the post-transcriptional regulation of gene expression. Furthermore, STAG3 localises to the centrosome independently of cohesin and interacts with proteins involved in mRNA localisation and stability. The knockdown of STAG3 in mESCs using siRNAs results in the destabilisation of the centrosome and the key P-body RNA-induced silencing complex (RISC) component TNRC6C, leading to the derepression of P-body localised mRNAs, such as DPPA3. Our results propose a model in which STAG3 collaborates with RNA-binding proteins (RBPs) and specific target mRNAs to control post-transcriptional gene expression and facilitate the transition from pluripotency in mESCs. Given that STAG3 is upregulated in various cancers, our results provide a novel perspective on how STAG proteins might contribute to cell identity and disease.

Supplementary Table 1. A)Patient details for Fresh Frozen test cohort, B) Patient details for validation cohort. Supplementary Figure 1. APOBEC mutation fraction of across multiple cancer types. Supplementary Figure 2. DNA methylation. Supplementary Figure 3. Candidate PeCa driver mutations. Supplementary Figure 4. Schematic of FAT1 is shown with locations of mutations and ethylation states of PeCa and normal penile tissue across the canonical FAT1 gene. Supplementary Figure 5. A) Example plot of Copy Number Alterations (CNA) in penile cancers, 450K methylation profiles for penile cancer and normal squamous epithelium, and output from GISTIC showing the frequency and significance of CNAs.

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). Commonly used approaches to study RNA silencing and RISC, such as those 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 AGO(2) 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, offers a route to the accurate and efficient generation of endogenous C-terminal tag fusions. Here, we used CRISPaint to generate the first reported recombinant AGO2 construct with a C-terminal tag: an endogenous C-terminal HaloTag fusion to AGO2 ( AGO2 HALO ) in human (A549) cells. We found that the AGO2 HALO fusion protein retains the capacity to interact with the key protein binding partner TNRC6A and that the C-terminal HaloTag does not affect cell viability. However, the AGO2 HALO fusion significantly impairs RNA cleavage and RNA silencing activity compared to control cells, and reduces nuclear localisation of the fusion protein. We conclude that the fusion of a C-terminal HaloTag to AGO2 is not appropriate for studying AGO2 and RISC. Our results stress the importance of fully validating recombinant tagging strategies to ensure that any results generated do not obscure critical functional defects.

It is now widely recognised that the tumour microenvironment (TME) is playing a crucial role in promoting tumour immune evasion by modulating the local immune infiltration. A growing body of evidence highlights the crucial role of the tumour extracellular matrix (ECM) in limiting trafficking of newly activated T cells in solid cancers, thus restricting their access to tumour and preventing the success of cancer immunotherapies. Accordingly, we are investigating whether targeting specific tumour ECM components may enhance T cell infiltration in triple-negative breast cancer (TNBC). Matrisome profiling of patient-derived fibroblasts and patient tumour tissues allowed us to identify two matrix proteins overexpressed in tumours and associated with T cell signatures, the matrix proteoglycan, versican (VCAN) and the glycoprotein, fibronectin (FN1). As there are currently no specific inhibitors of matrix proteins, we have modelled their loss-of-function using the clustered, regularly interspaced, short palindromic repeats-associated nuclease Cas9 (CRISPR-Cas9) technology with the goal of engineering cell-editable matrisomes which are enriched, reduced, or absent for specific matrix proteins. First, we constituted a panel of human and murine mammary fibroblast and TNBC cancer cell lines, which we have introduced with an inducible Cas9 protein expression. We measured VCAN and FN1 expression in the panel which showed distinct VCAN levels. Therefore, depending on VCAN basal levels, we have generated cell lines with VCAN overexpression, transient knockdown or stable knockout. Proteomic matrisome profiling of transient VCAN knockdown cells revealed that reducing VCAN may impact ECM abundance by decreasing VCAN direct binding partners. Finally, we used the edited cells to build 2D and 3D in vitro TME models to evaluate the impact of modulating VCAN expression on chimeric antigen receptor (CAR) T cell cytotoxicity and location. In parallel, we built in vivo mouse models of TNBC with controlled ECM deposition. Specifically, we have targeted mVcan and mFn1 in tumours and investigated the impact of their removal on tumour growth and immune cell infiltration. Overall, this work aims to identify immunomodulating features of the ECM which could be targeted to boost response to immunotherapies in solid cancers. Citation Format: Valentine Gauthier, Elly Tyler, Ying Liu, Tyson V. Sharp, Oliver MT Pearce. Targeting specific extracellular matrix proteins to reactivate T cell trafficking in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1339.

Genetic studies have identified several epithelial-derived genes associated with airway diseases. However, techniques used to study gene function frequently exceed the proliferative potential of primary human bronchial epithelial cells (HBECs) isolated from patients. Increased expression of the polycomb group protein BMI-1 extends the lifespan of HBECs while maintaining cell context plasticity. Herein we aimed to assess how BMI-1 expression impacted cellular functions and global mRNA expression. HBECs from six donors were transduced with lentivirus containing BMI-1 and cells were characterised, including by RNA sequencing and impedance measurement. BMI-1-expressing HBECs (B-HBECs) have a proliferative advantage and show comparable in vitro properties to low passage primary HBECs, including cell attachment/spreading and barrier formation. The B-HBEC mRNA signature was modestly different to HBECs, with only 293 genes differentially expressed (5% false discovery rate). Genes linked to epithelial mesenchymal transition and cell cycle were enriched in B-HBECs. We investigated the expression of genes implicated in asthma from genetic and expression studies and found that 97.6% of genes remained unaltered. We have shown that increased BMI-1 expression in HBECs delays lung epithelial cell senescence by promoting cell cycle progression and highlighted the flexible utility for B-HBECs as an important platform for studying airway epithelial mechanisms.

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 tumorigenesis via a number of different mechanisms. Approximately 45% of non-small cell lung cancers (NSCLC) are deficient in LIMD1, 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 −/− tumours in subcutaneous xenograft models, with no significant effect in LIMD1 +/+ cells. We have identified a novel drug tool with significant preclinical characterisation that serves as an excellent candidate to explore and define LIMD1-deficient cancers as a new therapeutic subgroup of critical unmet need.

- The miR-132/212 cluster controls ribosomal protein expression through directly targeting two transcriptional co-activators, BTAF1 and p300. Leishmania donovani -infected miR-132/212 -/- mice display increased IL-10 and reduced IFN g protein expression in Th1 cells, reduced hepatosplenomegaly, and increased parasite burdens. ABSTRACT 34 Determining the mechanisms that distinguish from 35 pathological chronic inflammation remains a fundamental challenge. miR-132 has 36 been shown to play largely immunoregulatory roles in immunity, however its role 37 in CD4 + T cell function is poorly understood. Here, we show that CD4 + T cells 38 express high levels of miR-132 and that T cell activation leads to miR-132 up- 39 regulation. The transcriptomic hallmark of splenic CD4 + T cells lacking the miR- 40 132/212 cluster during chronic infection is an increase in mRNAs levels of 41 ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR- 42 132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation 43 of RP transcription. Following infection with Leishmania donovani miR-132 -/- CD4 + 44 T cells display enhanced expression of IL-10 and decreased IFN g . This is 45 associated with reduced hepatosplenomegaly and enhanced pathogen load. The 46 enhanced IL-10 expression in miR-132 -/- Th1 cells is recapitulated in vitro 47 following treatment with phenylephrine, a drug reported to promote ribosome 48 synthesis. Our results uncover that miR-132/212-mediated regulation of RP 49 expression is critical for optimal CD4 + T cell activation and protective immunity 50 against pathogens.

Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR‐132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4+ T cell function is poorly understood. Here, we show that CD4+ T cells express high levels of miR‐132 and that T cell activation leads to miR‐132 up‐regulation. The transcriptomic hallmark of splenic CD4+ T cells lacking the miR‐132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co‐factor of B‐TFIID and novel miR‐132/212‐3p target, and p300 contribute towards miR‐132/212‐mediated regulation of RP transcription. Following infection with Leishmania donovani, miR‐132−/− CD4+ T cells display enhanced expression of IL‐10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL‐10 expression in miR‐132−/− Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR‐132/212‐mediated regulation of RP expression is critical for optimal CD4+ T cell activation and protective immunity against pathogens. The miR‐132/212 cluster plays important roles in acute inflammation and infection. This study shows that during pathogen‐induced chronic inflammation miR‐132‐mediated regulation of ribosomal protein expression is critical for CD4+ T cell activation and protective immunity. The miR‐132/212 cluster plays important roles in acute inflammation and infection. This study shows that during pathogen‐induced chronic inflammation miR‐132‐mediated regulation of ribosomal protein expression is critical for CD4+ T cell activation and protective immunity.

Primary cilia are sensory organelles involved in regulation of cellular signaling. Cilia loss is frequently observed in tumors; yet, the responsible mechanisms and consequences for tumorigenesis remain unclear. We demonstrate that cilia structure and function is disrupted in human pheochromocytomas – endocrine tumors of the adrenal medulla. This is concomitant with transcriptional changes within cilia-mediated signaling pathways that are associated with tumorigenesis generally and pheochromocytomas specifically. Importantly, cilia loss was most dramatic in patients with germline mutations in the pseudohypoxia-linked genes SDHx and VHL. Using a pheochromocytoma cell line derived from rat, we show that hypoxia and oncometabolite-induced pseudohypoxia are key drivers of cilia loss and identify that this is dependent on activation of an Aurora-A/HDAC6 cilia resorption pathway. We also show cilia loss drives dramatic transcriptional changes associated with proliferation and tumorigenesis. Our data provide evidence for primary cilia dysfunction contributing to pathogenesis of pheochromocytoma by a hypoxic/pseudohypoxic mechanism and implicates oncometabolites as ciliary regulators. This is important as pheochromocytomas can cause mortality by mechanisms including catecholamine production and malignant transformation, while hypoxia is a general feature of solid tumors. Moreover, pseudohypoxia-induced cilia resorption can be pharmacologically inhibited, suggesting potential for therapeutic intervention.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia‐inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF‐α and HIF‐β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour‐suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF‐α subunit. Here, we identify LIMD1 as a HIF‐1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF‐α degradation by modulating PHD2‐LIMD1‐VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF‐α protein degradation, inhibiting HIF‐1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1 This study identifies the tumour suppressor LIMD1 as a facilitator of an adaptive cellular response to hypoxia. Under hypoxic conditions, LIMD1 expression is induced, facilitating degradation of HIF, limiting HIF transcriptional activity and mitigating its pro‐tumorigenic effects. This study identifies the tumour suppressor LIMD1 as a facilitator of an adaptive cellular response to hypoxia. Under hypoxic conditions, LIMD1 expression is induced, facilitating degradation of HIF, limiting HIF transcriptional activity and mitigating its pro‐tumorigenic effects.

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-a and HIF-b subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumoursuppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-a subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-a degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-a protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers.