Breast cancer is the most common malignancy among women worldwide, causing >650,000 deaths annually. Despite advances in personalized therapies, many patients fail to obtain durable benefits from treatments tailored to their disease subtype. For instance, only some patients with PD-L1⁺ triple-negative breast cancer (TNBC) respond to chemotherapy plus immune checkpoint inhibitors. Moreover, while tumor-infiltrating lymphocyte levels correlate with improved survival in TNBC and HER2⁺ breast cancer, this is not true for HR⁺HER2- disease. Thus, other immunobiological features of the breast cancer microenvironment may hold clinically relevant prognostic or predictive value, especially in HR+HER2- tumors. Here, we investigated transcriptional signatures of immune cell infiltration across three public transcriptomic datasets from patients with breast cancer. The relative abundance and activation status of tumor-infiltrating mast cells were consistently associated with shifts in survival indicators amongst patients with HR+HER2- breast cancer. Specifically, infiltration by resting (but not activated) mast cells was associated with prolonged survival, correlating inversely with tumor infiltration by immune cells and proliferation markers in cancer cells, but positively with stromal richness. While based on retrospective transcriptional analyses, these findings suggest that interactions between mast cells and non-malignant components of the breast cancer microenvironment, particularly fibroblasts, may shape disease progression and treatment sensitivity.

Background: Resistance to CDK4/6 inhibitors (CDK4/6is) underlies treatment failure in patients with HR+HER2- breast cancer (BC) (Pandey et al., 2019). Thus, strategies breaking resistance to CDK4/6i+ET in women with HR+ BC are urgently awaited. Recent findings from Dr. Galluzzi in various immunocompetent mouse models of HR+ BC demonstrate that the CDK4/6i palbociclib (P) can be successfully combined with radiotherapy (RT) when delivered according to a precise treatment schedule (Petroni et al., 2021). These data inspired the initiation of a prospective, randomized phase II clinical trial comparing standard-of-care CDK4/6is plus ET vs RT followed by (à) CDK4/6is plus ET in patients with oligometastatic HR+ BC (NCT04563507).In this context, we set out to dissect the immunological mechanisms underlying sensitivity vs. resistance to treatment in HR+ BC exposed to P+ET vs. RTàP+ET. Methods: To dissect the impact of these treatments on HR+ BC, we performed scRNAseq on CD45+ cells infiltrating medroxyprogesterone acetate /7,12-Dimethylbenz[a]anthracene (MPA/DMBA)-driven carcinomas established in immunocompetent mice (a model of luminal B BC), bioinformatic analysis on public patient datasets, functional studies and efficacy studies. Results: We identified a hypoxia-inhibitable CCL2-depedent pathway recruiting IL17A+ γδ T cells to mouse HR+HER2- BCs after CDK4/6is, which repolarized tumor-associated macrophages (TAMs) towards an immunosuppressive CX3CR1+ phenotype. IL17A or γδ T cell signatures, as well as intratumoral γδ T cell or CX3CR1+ TAM abundance, correlated with tumor grade and reduced survival in two cohorts of HR+HER2- BC patients. Consistent with mouse data, circulating γδ T cells and plasma CCL2 levels negatively correlated with progression-free survival (PFS) in two series of HR+HER2- BC patients receiving CDK4/6is. Moreover, intratumoral γδ T cells were increased in HR+HER2- BC biopsies upon relapse on CDK4/6is compared to paired baseline biopsies. CX3CR1+ TAMs had negative prognostic impact in patients with HR+HER2- BC treated with anti-PD-1 and RT. Conclusions: Our observations suggest that γδ T cells and CX3CR1+ TAMs may favor resistance to CDK4/6is in patients with HR+HER2- BC, and hence constitute potential targets to delay disease progression. Citation Format: Claudia Galassi, Giulia Petroni, Lorenzo Galluzzi. An immunological mechanism of resistance to CDK4/6 inhibitors in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-04-18.

Mitochondrial permeability transition (MPT)-driven necrosis and necroptosis are regulated variants of cell death that can drive inflammation or even promote antigen-specific immune responses. In oncological settings, indolent inflammatory reactions have been consistently associated with accelerated disease progression and resistance to treatment. Conversely, adaptive immune responses specific for tumor-associated antigens are generally restraining tumor development and contribute to treatment sensitivity. Here, we harnessed female C57BL/6J mice lacking key regulators of MPT-driven necrosis and necroptosis to investigate whether whole-body defects in these pathways would influence mammary carcinogenesis as driven by subcutaneous slow-release medroxyprogesterone acetate (MPA, M) pellets plus orally administered 7,12-dimethylbenz[a]anthracene (DMBA, D), an in vivo model that recapitulates multiple facets of the biology and immunology of human hormone receptor positive (HR+) breast cancer. Our data demonstrate that female mice bearing a whole-body, homozygous deletion in peptidylprolyl isomerase F (Ppif), which encodes a key regulator of MPT-driven necrosis commonly known as CYPD, but not female mice with systemic defects in necroptosis as imposed by the whole body-deletion homozygous of receptor-interacting serine-threonine kinase 3 (Ripk3) or mixed lineage kinase domain like pseudokinase (Mlkl), are more susceptible to M/D-driven carcinogenesis than their wild-type counterparts. These findings point to CYPD as to an oncosuppressive protein that restrains HR+ mammary carcinogenesis in mice, at least potentially via MPT-driven necrosis.

CDK4/6 inhibitors are central to the clinical management of HR+HER2- breast cancer. We have recently demonstrated that immunosuppressive, IL17-secreting γδ T cells recruited to the tumor microenvironment by a CCL2-dependent mechanism upon CDK4/6 inhibition can repolarize tumor-associated macrophages toward a CX3CR1+ phenotype associated with resistance to therapy.

Resistance to cyclin-dependent kinase 4/6 (CDK4/CDK6) inhibitors leads to treatment failure and disease progression in women with hormone receptor+HER2− (HR+HER2−) breast cancer (BC). We delineated a hypoxia-sensitive, CCL2-dependent pathway recruiting interleukin-17A (IL-17A)-secreting γδ T cells to mouse HR+HER2− BCs following CDK4/CDK6 inhibition, resulting in repolarization of tumor-associated macrophages (TAMs) toward an immunosuppressive CX3CR1+ phenotype associated with resistance. Increased IL-17A signaling and intratumoral γδ T cell abundance positively correlated with advanced grade and/or reduced survival in two cohorts of individuals with HR+HER2− BC. Circulating γδ T cells and plasma CCL2 levels negatively correlated with progression in an independent series of individuals with HR+HER2− BC receiving CDK4/CDK6 inhibitors. Intratumoral γδ T cells were increased in post- versus pretreatment biopsies from individuals with HR+HER2− BC relapsing on CDK4/CDK6 inhibitors. CX3CR1+ TAMs had negative prognostic impact in women with HR+HER2− BC receiving neoadjuvant PD-1 blockage and radiotherapy. Thus, γδ T cells and CX3XR1+ TAMs may favor resistance to CDK4/CDK6 inhibitors in individuals with HR+HER2− BC. Petroni et al. report that the infiltration of IL-17A-secreting γδ T cells in the tumor microenvironment coupled with the accumulation of immunosuppressive macrophages is associated with resistance to CDK4/CDK6 inhibition in HR+HER2− breast cancer.

Estrogen receptor (ER)+ breast malignancies are poorly infiltrated by immune cells, hence exhibiting limited sensitivity to immune checkpoint inhibitors (ICIs). Recent data from Palomeque et al. demonstrate that ER signaling actively contributes to such an immunoevasive phenotype by preventing the nuclear factor LCOR from establishing an ICI-sensitive tumor microenvironment.

γδ T cells, a heterogeneous lymphocyte subset operating at the interface between innate and adaptive immunity, mediate ambivalent and context-dependent effects in oncological settings. Recent work by Rozalén et al, (2025) demonstrates that IL-17-secreting γδ T cells favor the outgrowth of TIM-3+ triple-negative breast cancer metastases by establishing local immunosuppression. Recent data report a role for IL-17-secreting γδ T cells in facilitating the establishment of TIM-3+ TNBC metastases via local immunosuppression.

Background: Poly (ADP-ribose) polymerase inhibitors (PARPis) lead to synthetic lethality when used in cancers with homologous recombination deficiency (HRD). However, the development of resistance to PARPis is a recurrent problem thus limits the duration of response and hence the clinical utility of these agents. Here, we describe the antineoplastic and immunomodulatory effects of OX425, a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy, resulting in constitutive PARP1 hyperactivation and consequent exhaustion of the DNA damage response. Methods: OX425-induced PARP trapping, hyperactivation and cell cytotoxicity were examined in vitro in HRD and homologous recombination proficient (HRP) human cancer cells, as well as in non-transformed cell lines. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. OX425 effects on the innate and adaptive immune responses were assessed by following STING activation and T-cell mediated anti-tumor cytotoxicity. RNAseq analysis in HRP/HRD tumor cells treated with OX425 or PARP inhibitors was employed to uncover the molecular mechanisms underlying OX425 effects. The anticancer efficacy of OX425 was assessed in vivo in different HRD and HRP tumor models. OX425-induced PARP activation and tumor infiltration by immune cells were analyzed by flow cytometry. Results: At odds with conventional PARP inhibitors, OX425 bound to and hyperactivated PARP1 with high affinity in a dose-dependent manner, resulting in elevated cytotoxicity to multiple cancer cells (breast, ovarian, prostate, colon, hematological, endometrial cancers) irrespective of HR status. Interestingly, long-term treatment with OX425 did not show any mutagenicity compared to PARPi. The activity of OX425 was specific to tumor cells, as no significant effect on cell viability was observed for normal cells, at odds with PARP inhibitors. In line with in vitro results, OX425 mediated considerable anticancer effects in vivo. Moreover, OX425 triggered activation of the STING pathway and CCL5 secretion in the EMT6 mouse mammary carcinoma model. The anticancer effect of OX425 was coupled with tumor-targeting T cell responses. In MPA/DMBA-driven mammary tumors, OX425 mediated considerable anticancer effects in monotherapy and synergistic effects in combination with PD1 inhibition. Moreover, OX425 treatment significantly delayed acquired resistance to olaparib in BRCA1 mutated MDA-MB-436 cell-derived xenografts. Conclusions: Our results provide preclinical rationale for using OX425 to trigger DNA damage exhaustion and STING activation in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD or HRP tumors Citation Format: Vlada Zakharova, Claudia Galassi, Chloé Doizelet, Vincent Hayes, Lorenzo Galluzzi, Wael Jdey. PARP1 hyperactivation by the decoy oligodeoxynucleotide OX425 mediates DNA repair abrogation and unleashes the anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6200.