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Overexpression of the HER2 protein in breast cancer patients is a predictor of poor prognosis and resistance to therapies. We used an inducible breast cancer transformation system that allows investigation of early molecular changes. HER2 overexpression to similar levels as those observed in a subtype of HER2-positive breast cancer patients induced transformation of MCF10A cells and resulted in gross morphological changes, increased anchorage-independent growth of cells, and altered the transcriptional programme of genes associated with oncogenic transformation. Global phosphoproteomic analysis during HER2 induction predominantly detected an increase in protein phosphorylation. Intriguingly, this correlated with chromatin opening, as measured by ATAC-seq on acini isolated from 3D cell culture. HER2 overexpression resulted in opening of many distal regulatory regions and promoted reprogramming-associated heterogeneity. We found that a subset of cells acquired a dedifferentiated breast stem-like phenotype, making them likely candidates for malignant transformation. Our data show that this population of cells, which counterintuitively enriches for relatively low HER2 protein abundance and increased chromatin accessibility, possesses transformational drive, resulting in increased anchorage-independent growth in vitro compared to cells not displaying a stem-like phenotype.

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 IFNγ-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.Competing Interest StatementThe authors have declared no competing interest.