STING-related biomarkers in BC PDXs

PARP inhibitors (PARPi) are effective in tumors with homologous recombination repair (HRR) deficiency (HRD), typically identified by germline/tumor mutations. However, genetic testing may miss intrinsic PARPi sensitivity and resistance. We evaluated strategies to improve detection and longitudinal monitoring of HRD across >500 tumor samples and >20 paired liquid biopsies, integrating genetic, genomic, and functional readouts. HRD was more frequent in high-grade ovarian cancer (HGOC; 52%) than in metastatic breast (mBC; 12%) or prostate cancer (mPC; 20%). Assay concordance was low-to-moderate, underscoring complementarity. RAD51 testing and the genomic instability score identified HRD in tumors lacking pathogenic HRR mutations (6% and 30% in mBC, 38% and 46% in HGOC, 14% and 27% in mPC, respectively). Longitudinal ctDNA sequencing revealed BRCA1/BRCA2 reversion mutations in >30% of post-PARPi mBC samples, which were associated with poor response to subsequent platinum therapy. These findings support the use of complementary HRD biomarkers in tissue and liquid biopsy to guide PARPi use and monitor response.

MDA single cells annotation in cell types

Up-regulation of Interferon Stimulated Genes (ISGs) in PARPi-sensitive PDX models

Bulk RNA sequencing of Mouse Derived Allograft (MDA) tumors.

Advances in breast cancer (BC) therapy are limited by the absence of well-established biomarkers for DNA-damage targeted treatments. We evaluated the predictive and prognostic value of homologous recombination repair (HRR) deficiency (HRD) by RAD51 nuclear foci and stromal tumour-infiltrating lymphocytes (TILs) in early-stage BC patients with suspected germline susceptibility. Among 291 patients, HRD by RAD51 was found in 78.4% of tumours, and 69.8% had low TILs (<30%). In 178 patients treated with neoadjuvant chemotherapy, pathologic complete response (pCR) was higher in those with HRD vs HRR-proficient (HRP) tumours (52.3% vs 36.4%); RAD51 remained independently associated with pCR (p = 0.03). Overall survival (OS) favoured HRD, with 5-year OS of 89.2% vs 82.8% in HRP (p = 0.009), with stronger evidence in triple-negative TILs-low disease (p = 0.005). These findings support RAD51-based HRD assessment as a predictive and prognostic biomarker that may guide treatment decisions in early-stage BC.

Interferon responses up-regulation in MDA tumor microenvironment

Bulk RNA sequencing of PDX230OR8 tumors

The co-occurrence of germline and somatic oncogenic alterations is frequently observed in breast cancer, yet their combined influence on tumour evolution and therapy resistance remains poorly defined. Through an integrated clinicogenomic analysis of more than 5,800 patients, we show that germline (g) pathogenic variants dictate the evolutionary trajectory of acquired resistance. We specifically find that gBRCA2-associated tumours are uniquely predisposed to develop acquired RB1 loss-of-function alterations, resulting in poor outcomes on standard-of-care frontline CDK4/6 inhibitor (CDK4/6i) combinations. This vulnerability is driven by a dual mechanism: baseline RB1 hemizygosity (heterozygous loss resulting in a single functional RB1 allele), which lowers the evolutionary barrier to biallelic inactivation, and ongoing homologous recombination deficiency, which promotes acquisition of RB1 loss-of-function alterations under the selective pressure of CDK4/6i. Preclinical models from gBRCA2 carriers showed near-uniform resistance to CDK4/6i, with consistent post-treatment Rb loss. Across multiple independent models and in our clinical data, PARP inhibition consistently outperformed CDK4/6i. Our findings suggest that prioritizing PARP inhibition in gBRCA2 carriers may intercept RB1-loss trajectories and delay resistance. More broadly, we establish a predictive framework for forecasting drug-resistant trajectories based on pre-treatment allelic configuration and mutational signatures.

Abstract Introduction: BRCA1 is a tumor suppressor required for homologous recombination repair (HRR) of DNA double-strand breaks. BRCA1 loss-of-function mutations cause HRR deficiency and sensitize tumors to platinum-based chemotherapy and PARP inhibitors (PARPi). Standard gene-level testing may miss other mechanisms affecting BRCA1 protein functionality, such as BRCA1 promoter methylation, or other PARPi response molecular determinants, such as hypomorphic protein expression that partially restores HRR, leading to PARPi resistance. Current standard assays suitable for formalin-fixed paraffin-embedded (FFPE) tissue, such as immunohistochemistry, are limited in their ability to characterize BRCA1 protein levels - highlighting the need for new approaches for protein-level quantification. Methods: Targeted proteomics was performed on an Orbitrap Exploris 480 mass spectrometer coupled to an Evosep One nanoLC to quantify BRCA1 peptides spanning the N-terminal RING domain, Exon 11 and C-terminal BRCT domain, enabling BRCA1 hypomorph detection based on domain-specific peptide abundance. The targeted panel assay also included additional DNA repair proteins. This assay was applied to 56 FFPE tumors from 26 patient-derived xenografts (PDX), including 7 samples collected at acquired olaparib resistance. Results: PDX tumours from BRCA1 wild-type, BRCA2-mutant, or PALB2-mutant models (n=10), had a median BRCA1 peptide expression level of 45.8 amol/µg (range 20.7-120.9), consistent with a normal-expression reference range. In contrast, PDX tumors with BRCA1 promoter hypermethylation (n=4) exhibited low BRCA1 peptide levels (< 1.8 amol/µg). At acquired olaparib resistance, tumours from the same models showed a ∼25-fold BRCA1 protein increase, consistent with demethylation and restored BRCA1 expression. Among BRCA1-mutant PDX models (n=15), most models exhibited low expression, consistent with loss-of-function mutations (n=8 with < 5 amol/µg). Targeted proteomics further identified seven candidate BRCA1-mutant PDX hypomorphic cases in which at least one BRCA1 domain peptide exceeded the ∼5 amol/µg threshold; their BRCA1 peptide levels ranged from 6.2 to 31.7 amol/µg. Six out of seven hypomorph cases (86%) were PARPi resistant. Low 53BP1 expression was observed in four PARPi-resistant PDX models; three of these had known 53BP1 dysregulation or mutation. However, one model showed low 53BP1 despite the absence of a known TP53BP1 mutation, implicating 53BP1 loss as a plausible contributor to resistance. Conclusion: These data support this targeted proteomic assay as a practical approach to distinguish normal from low BRCA1 expression at the protein level, nominate hypomorphic BRCA1 variants linked to PARPi resistance, and identify deficiency in additional resistance biomarkers. Citation Format: Beom-Jun Kim, Alba Llop-Guevara, Steve Sweet, Camille Lombard-Banek, Robert Hanson, Chris Richardson, Violeta Serra, Elizabeth Harrington, Josep Forment, Yeoun Jin Kim. Absolute quantification by targeted proteomics identifies BRCA1 hypomorphs and other PARP inhibitor resistance mechanisms in patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7689.

Combination of PARPi and STINGa in vivo

Same HRR functionality, PARP enzymatic inhibition and DNA damage in different mouse strains

PARPi affects the peripheral immune composition in patients

Image analysis of tissue cyclic immunofluorescence in HRR-altered patients

Coupled scRNA-seq and scTCR-seq in peripheral blood identified hyperexpanded TCR clonotypes in the CD8 cytotoxic and NK-like T cells subsets

Inflammation is a biological phenomenon beneficial for homeostasis, but it is unfavorable if dysregulated. Although major progress has been made in characterizing inflammation in specific diseases, a global, holistic understanding is still elusive. This is particularly intriguing, considering its function for human health and the potential for modern medicine if fully deciphered. In this study, we leveraged advances in single-cell transcriptomics to delineate inflammatory processes of circulating immune cells during infection, immune-mediated inflammatory diseases and cancer. Our single-cell atlas of more than 6.5 million peripheral blood mononuclear cells from 1,047 patients (56% female, 43% male) and 19 diseases allowed us to learn a comprehensive model of inflammation in circulating immune cells. The atlas expands current knowledge of the biology of inflammation of immune-mediated diseases, acute and chronic inflammatory diseases, infections and solid tumors and lays the foundation to develop a disease classification framework using unsupervised as well as explainable machine learning. Beyond a disease-centered analysis, we charted altered activity of inflammatory molecules in peripheral blood cells, depicting discriminative inflammation-related genes to further understand mechanisms of inflammation. We present a rich resource for the community and lay the groundwork for learning a classifier for inflammatory diseases, presenting cells in circulation as living biomarkers.

The androgen receptor (AR) is expressed in 75% of estrogen receptor-positive (ER+) breast cancers (BC). Selective AR modulators (SARMs), like EP0062, present a promising therapeutic strategy for ER + BC, particularly in patients who cannot tolerate endocrine therapy (ET) or whose tumors have developed resistance. We aimed to study the antitumor activity of EP0062 in ER+ patient-derived xenograft (PDX) models. EP0062 displayed comparable antitumor efficacy to selective ER degraders (SERDs), including in PDXs with ESR1, PIK3CA, or PTEN mutations. Tumors sensitive to SARMs were enriched in GATA3 mutations. EP0062 treatment induced AR-target genes across all models tested. A transcriptional signature associated with SARM sensitivity was identified, primarily driven by proliferation-related processes, consistent with a significant decrease in S-phase cell cycle proteins upon treatment in EP0062-sensitive models. In some EP0062-resistant tumors, the combination with palbociclib enhanced the antitumor effect of EP0062, suggesting a potential strategy for metastatic patients with acquired ET resistance.

Natural Killer (NK) cells depletion in vivo

Analysis of the mechanism of PARPi resistance in MDA594ORs