Targeting the PREX2/RAC1/PI3Kβ Signaling Axis Confers Sensitivity to Clinically Relevant Therapeutic Approaches in Melanoma.

Metastatic melanoma remains a major clinical challenge. Large-scale genomic sequencing of melanoma has identified bona fide activating mutations in RAC1, which are associated with resistance to BRAF-targeting therapies. Targeting the RAC1-GTPase pathway, including the upstream activator PREX2 and the downstream effector PI3Kβ, could be a potential strategy for overcoming therapeutic resistance, limiting melanoma recurrence, and suppressing metastatic progression.

High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS.

The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target.

Spatial multiplex analysis of lung cancer reveals that regulatory T cells attenuate KRAS-G12C inhibitor-induced immune responses.

Kirsten rat sarcoma virus (KRAS)-G12C inhibition causes remodeling of the lung tumor immune microenvironment and synergistic responses to anti-PD-1 treatment, but only in T cell infiltrated tumors. To investigate mechanisms that restrain combination immunotherapy sensitivity in immune-excluded tumors, we used imaging mass cytometry to explore cellular distribution in an immune-evasive KRAS mutant lung cancer model. Cellular spatial pattern characterization revealed a community where CD4 and CD8 T cells and dendritic cells were gathered, suggesting localized T cell activation.

Combining RAS(ON) G12C-selective inhibitor with SHP2 inhibition sensitises lung tumours to immune checkpoint blockade.

Mutant selective drugs targeting the inactive, GDP-bound form of KRAS have been approved for use in lung cancer, but resistance develops rapidly. Here we use an inhibitor, (RMC-4998) that targets RAS in its active, GTP-bound form, to treat KRAS mutant lung cancer in various immune competent mouse models. RAS pathway reactivation after RMC-4998 treatment could be delayed using combined treatment with a SHP2 inhibitor, which not only impacts tumour cell RAS signalling but also remodels the tumour microenvironment to be less immunosuppressive.

Deep cell phenotyping and spatial analysis of multiplexed imaging with TRACERx-PHLEX.

The growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations.

Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone.

Palmitoyl transferase ZDHHC20 promotes pancreatic cancer metastasis.

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system.

CRISPR-Cas9 Screening Identifies KRAS-Induced COX2 as a Driver of Immunotherapy Resistance in Lung Cancer.

Oncogenic KRAS impairs antitumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of the mechanisms by which oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive COX2 in cancer cells.

Spatial Architecture of Myeloid and T Cells Orchestrates Immune Evasion and Clinical Outcome in Lung Cancer.

Unlabelled: Understanding the role of the tumor microenvironment (TME) in lung cancer is critical to improving patient outcomes. We identified four histology-independent archetype TMEs in treatment-naïve early-stage lung cancer using imaging mass cytometry in the TRACERx study (n = 81 patients/198 samples/2.3 million cells).

Exploiting the therapeutic implications of KRAS inhibition on tumor immunity.

Over the past decade, RAS oncogenic proteins have transitioned from being deemed undruggable to having two clinically approved drugs, with several more in advanced stages of development. Despite the initial benefit of KRAS-G12C inhibitors for patients with tumors harboring this mutation, the rapid emergence of drug resistance underscores the urgent need to synergize these inhibitors with other therapeutic approaches to improve outcomes. RAS mutant tumor cells can create an immunosuppressive tumor microenvironment (TME), suggesting an increased susceptibility to immunotherapies following RAS inhibition.

LKB1 depletion-mediated epithelial-mesenchymal transition induces fibroblast activation in lung fibrosis.

The factors that determine fibrosis progression or normal tissue repair are largely unknown. We previously demonstrated that autophagy inhibition-mediated epithelial-mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments local myofibroblast differentiation in pulmonary fibrosis by paracrine signalling. Here, we report that liver kinase B1 (LKB1) inactivation in ATII cells inhibits autophagy and induces EMT as a consequence.

A palmitoyl transferase chemical-genetic system to map ZDHHC-specific S-acylation.

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs.

Facts and Hopes on RAS Inhibitors and Cancer Immunotherapy.

Although the past decade has seen great strides in the development of immunotherapies that reactivate the immune system against tumors, there have also been major advances in the discovery of drugs blocking oncogenic drivers of cancer growth. However, there has been very little progress in combining immunotherapies with drugs that target oncogenic driver pathways. Some of the most important oncogenes in human cancer encode RAS family proteins, although these have proven challenging to target.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS). Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma.

Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development. Here we propose that environmental particulate matter measuring ≤2.

Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.

P110α is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110α by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110α/RAS interaction in tumour initiation and growth.

Micro-CT acquisition and image processing to track and characterize pulmonary nodules in mice.

X-ray computed tomography is a reliable technique for the detection and longitudinal monitoring of pulmonary nodules. In preclinical stages of diagnostic or therapeutic development, the miniaturized versions of the clinical computed tomography scanners are ideally suited for carrying out translationally-relevant research in conditions that closely mimic those found in the clinic. In this Protocol, we provide image acquisition parameters optimized for low radiation dose, high-resolution and high-throughput computed tomography imaging using three commercially available micro-computed tomography scanners, together with a detailed description of the image analysis tools required to identify a variety of lung tumor types, characterized by specific radiological features.

RAS oncogenic activity predicts response to chemotherapy and outcome in lung adenocarcinoma.

Activating mutations in KRAS occur in 32% of lung adenocarcinomas (LUAD). Despite leading to aggressive disease and resistance to therapy in preclinical studies, the KRAS mutation does not predict patient outcome or response to treatment, presumably due to additional events modulating RAS pathways. To obtain a broader measure of RAS pathway activation, we developed RAS84, a transcriptional signature optimised to capture RAS oncogenic activity in LUAD.

An Immunogenic Model of KRAS-Mutant Lung Cancer Enables Evaluation of Targeted Therapy and Immunotherapy Combinations.

Unlabelled: Mutations in oncogenes such as KRAS and EGFR cause a high proportion of lung cancers. Drugs targeting these proteins cause tumor regression but ultimately fail to elicit cures. As a result, there is an intense interest in how to best combine targeted therapies with other treatments, such as immunotherapies.

Therapeutic KRAS inhibition drives effective interferon-mediated antitumor immunity in immunogenic lung cancers.

Recently developed KRAS inhibitory drugs are beneficial to lung cancer patients harboring KRAS mutations, but drug resistance frequently develops. Because of the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, these drugs can indirectly affect antitumor immunity, providing a rationale for their combination with immune checkpoint blockade. In this study, we have characterized how KRAS inhibition reverses immunosuppression driven by oncogenic KRAS in a number of preclinical lung cancer models with varying levels of immunogenicity.