Epithelial ovarian cancer (EOC) represents the most lethal gynaecological malignancy, yet understanding the connections between its molecular subtypes and their therapeutic implications remains incomplete. We conducted mass spectrometry-based proteomics analyses of 154 EOC tumour samples and 29 normal fallopian tubes, and single-cell RNA sequencing (scRNA-seq) analyses of an additional eight EOC tumours to classify proteomic subtypes and assess their cellular ecosystems and clinical significance. The efficacy of identified therapeutic targets was evaluated in patient-derived xenograft (PDX) and orthotopic mouse models. We identified four proteomic subtypes with distinct clinical relevance: malignant proliferative (C1), immune infiltrating (C2), Fallopian-like (C3) and differentiated (C4) subtypes. C2 subtype was characterized by lymphocyte infiltration, notably an increased presence of GZMK CD8+ T cells and phagocytosis-like MRC+ macrophages. Additionally, we identified CD40 as a specific prognostic factor for C2 subtype. The interaction between CD40+ phagocytosis-like macrophages and CD40RL+ IL17R CD4+ T cells was correlated with a favourable prognosis. Finally, we established a druggable landscape for non-immune EOC patients and verified a TYMP inhibitor as a promising therapeutic strategy. Our study refines the current immune subtype for EOC, highlighting CD40 agonists as promising therapies for C2 subtype patients and targeting TYMP for non-immune patients.

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.

Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines in vitro and tumor development in vivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured T cells in vitro, compromises anti-tumor immunity in vivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients.