Predicting cancer-specific vulnerability via data-driven detection of synthetic lethality.

Synthetic lethality occurs when the inhibition of two genes is lethal while the inhibition of each single gene is not. It can be harnessed to selectively treat cancer by identifying inactive genes in a given cancer and targeting their synthetic lethal (SL) partners. We present a data-driven computational pipeline for the genome-wide identification of SL interactions in cancer by analyzing large volumes of cancer genomic data. First, we show that the approach successfully captures known SL partners of tumor suppressors and oncogenes. We then validate SL predictions obtained for the tumor suppressor VHL. Next, we construct a genome-wide network of SL interactions in cancer and demonstrate its value in predicting gene essentiality and clinical prognosis. Finally, we identify synthetic lethality arising from gene overactivation and use it to predict drug efficacy. These results form a computational basis for exploiting synthetic lethality to uncover cancer-specific susceptibilities.

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression.

Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.

PTEN deficiency exposes a requirement for an ARF GTPase module for integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

A 'synthetic-sickness' screen for senescence re-engagement targets in mutant cancer backgrounds.

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAßGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments.

Multi-scale in vivo imaging of tumour development using a germline conditional triple-reporter system.

Imaging reporter genes are indispensable for visualising biological processes in living subjects, particularly in cancer research where they have been used to observe tumour development, cancer cell dissemination, and treatment response. Engineering reporter genes into the germline frequently involves single imaging modality reporters operating over limited spatial scales. To address these limitations, we developed an inducible triple-reporter mouse model (Rosa26LSL - NRL) that integrates reporters for complementary imaging modalities, flfluorescence, bioluminescence and positron emission tomography (PET), along with inducible Cre-lox functionality for precise spatiotemporal control of reporter expression. We demonstrated robust reporter inducibility across various tissues in the Rosa26LSL - NRL mouse, facilitating effective tracking and characterisation of tumours in liver and lung cancer mouse models. We precisely pinpointed tumour location using multimodal whole-body imaging which guided in situ lung microscopy to visualise cell-cell interactions within the tumour microenvironment. The triple-reporter system establishes a robust new platform technology for multi-scale investigation of biological processes within whole animals, enabling tissue-specific and sensitive cell tracking, spanning from the whole-body to cellular scales.

Scar/WAVE drives actin protrusions independently of its VCA domain using proline-rich domains.

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. Fine-tuning of this process is critical to ensure new actin filaments are formed only at specific times and in defined regions of the cell. The Scar/WAVE complex is the main catalyst of pseudopod and lamellipodium formation during cell migration. It is a pentameric complex highly conserved through eukaryotic evolution and composed of Scar/WAVE, Abi, Nap1/NCKAP1, Pir121/CYFIP, and HSPC300/Brk1. Its function is usually attributed to activation of the Arp2/3 complex through Scar/WAVE's VCA domain, while other parts of the complex are expected to mediate spatial-temporal regulation and have no direct role in actin polymerization. Here, we show in both B16-F1 mouse melanoma and Dictyostelium discoideum cells that Scar/WAVE without its VCA domain still induces the formation of morphologically normal, actin-rich protrusions, extending at comparable speeds despite a drastic reduction of Arp2/3 recruitment. However, the proline-rich regions in Scar/WAVE and Abi subunits are essential, though either is sufficient for the generation of actin protrusions in B16-F1 cells. We further demonstrate that N-WASP can compensate for the absence of Scar/WAVE's VCA domain and induce lamellipodia formation, but it still requires an intact WAVE complex, even if without its VCA domain. We conclude that the Scar/WAVE complex does more than directly activating Arp2/3, with proline-rich domains playing a central role in promoting actin protrusions. This implies a broader function for the Scar/WAVE complex, concentrating and simultaneously activating many actin-regulating proteins as a lamellipodium-producing core.

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function.

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.

The small GTPase ARF3 controls invasion modality and metastasis by regulating N-cadherin levels.

ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis.

Phenotypic profiling of solute carriers characterizes serine transport in cancer.

Serine is a vital amino acid in tumorigenesis. While cells can perform de novo serine synthesis, most transformed cells rely on serine uptake to meet their increased biosynthetic requirements. Solute carriers (SLCs), a family of transmembrane nutrient transport proteins, are the gatekeepers of amino acid acquisition and exchange in mammalian cells and are emerging as anticancer therapeutic targets; however, the SLCs that mediate serine transport in cancer cells remain unknown. Here we perform an arrayed RNAi screen of SLC-encoding genes while monitoring amino acid consumption and cell proliferation in colorectal cancer cells using metabolomics and high-throughput imaging. We identify SLC6A14 and SLC25A15 as major cytoplasmic and mitochondrial serine transporters, respectively. We also observe that SLC12A4 facilitates serine uptake. Dual targeting of SLC6A14 and either SLC25A15 or SLC12A4 diminishes serine uptake and growth of colorectal cancer cells in vitro and in vivo, particularly in cells with compromised de novo serine biosynthesis. Our results provide insight into the mechanisms that contribute to serine uptake and intracellular handling.

Omentum-derived matrix enables the study of metastatic ovarian cancer and stromal cell functions in a physiologically relevant environment.

High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and in vitro models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients. Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma.

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53-/- null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.

MICAL1 regulates actin cytoskeleton organization, directional cell migration and the growth of human breast cancer cells as orthotopic xenograft tumours.

The Molecule Interacting with CasL 1 (MICAL1) monooxygenase has emerged as an important regulator of cytoskeleton organization via actin oxidation. Although filamentous actin (F-actin) increases MICAL1 monooxygenase activity, hydrogen peroxide (H2O2) is also generated in the absence of F-actin, suggesting that diffusible H2O2 might have additional functions. MICAL1 gene disruption by CRISPR/Cas9 in MDA MB 231 human breast cancer cells knocked out (KO) protein expression, which affected F-actin organization, cell size and motility. Transcriptomic profiling revealed that MICAL1 deletion significantly affected the expression of over 700 genes, with the majority being reduced in their expression levels. In addition, the absolute magnitudes of reduced gene expression were significantly greater than the magnitudes of increased gene expression. Gene set enrichment analysis (GSEA) identified receptor regulator activity as the most significant negatively enriched molecular function gene set. The prominent influence exerted by MICAL1 on F-actin structures was also associated with changes in the expression of several serum-response factor (SRF) regulated genes in KO cells. Moreover, MICAL1 disruption attenuated breast cancer tumour growth in vivo. Elevated MICAL1 gene expression was observed in invasive breast cancer samples from human patients relative to normal tissue, while MICAL1 amplification or point mutations were associated with reduced progression free survival. Collectively, these results demonstrate that MICAL1 gene disruption altered cytoskeleton organization, cell morphology and migration, gene expression, and impaired tumour growth in an orthotopic in vivo breast cancer model, suggesting that pharmacological MICAL1 inhibition could have therapeutic benefits for cancer patients.

Selection of established tumour cells through narrow diameter micropores enriches for elevated Ras/Raf/MEK/ERK MAPK signalling and enhanced tumour growth.

As normal cells become cancer cells, and progress towards malignancy, they become progressively softer. Advantages of this change are that tumour cells become more deformable, and better able to move through narrow constraints. We designed a positive selection strategy that enriched for cells which could move through narrow diameter micropores to identify cell phenotypes that enabled constrained migration. Using human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, we found that micropore selection favoured cells with relatively higher Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signalling, which affected actin cytoskeleton organization, focal adhesion density and cell elasticity. In this follow-up study, we provide further evidence that selection through micropores enriched for cells with altered cell morphology and adhesion. Additional analysis of RNA sequencing data revealed a set of transcripts associated with small cell size that was independent of constrained migration. Gene set enrichment analysis identified the 'matrisome' as the most significantly altered gene set linked with small size. When grown as orthotopic xenograft tumours in immunocompromised mice, micropore selected cells grew significantly faster than Parent or Flow-Sorted cells. Using mathematical modelling, we determined that there is an interaction between 1) the cell to gap size ratio; 2) the bending rigidity of the cell, which enable movement through narrow gaps. These results extend our previous conclusion that Ras/Raf/MEK/ERK MAPK signalling has a significant role in regulating cell biomechanics by showing that the selective pressure of movement through narrow gaps also enriches for increased tumour growth in vivo.

Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression.

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism.

An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

BRD4-mediated repression of p53 is a target for combination therapy in AML.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi's ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

Novel beta-propeller of the BTB-Kelch protein Krp1 provides a binding site for Lasp-1 that is necessary for pseudopodial extension.

Kelch-related protein 1 (Krp1) is up-regulated in oncogene-transformed fibroblasts. The Kelch repeats interact directly with the actin-binding protein Lasp-1 in membrane ruffles at the tips of pseudopodia, where both proteins are necessary for pseudopodial elongation. Herein, we investigate the molecular basis for this interaction. Probing an array of overlapping decapeptides of Rattus norvegicus (Rat) Krp1 with recombinant Lasp-1 revealed two binding sites; one ((317)YDPMENECYLT(327)) precedes the first of five Kelch repeats, and the other ((563)TEVNDIWKYEDD(574)) is in the last of the five Kelch repeats. Mutational analysis established that both binding sites are necessary for Krp1-Lasp-1 interaction in vitro and function in vivo. The crystal structure of the C-terminal domain of rat Krp1 (amino acids 289-606) reveals that both binding sites are brought into close proximity by the formation of a novel six-bladed beta-propeller, where the first blade is not formed by a Kelch repeat.