Combinatorial clinical trials of PARP inhibitors with immunotherapies are ongoing, yet the immunomodulatory effects of PARP inhibition have been incompletely studied. Here, we sought to dissect the mechanisms underlying PARP inhibitor-induced changes in the tumor microenvironment of BRCA1-defi cient triple-negative breast cancer (TNBC). We demonstrate that the PARP inhibitor olaparib induces CD8 + T-cell infi ltration and activation in vivo , and that CD8 + T-cell depletion severely compromises antitumor effi cacy. Olaparib-induced T-cell recruitment is mediated through activation of the cGAS/STING pathway in tumor cells with paracrine activation of dendritic cells and is more pronounced in HR-defi cient compared with HR-profi cient TNBC cells and in vivo models. CRISPR-mediated knockout of STING in cancer cells prevents proinfl ammatory signaling and is suffi cient to abolish olaparib-induced T-cell infi ltration in vivo. These fi ndings elucidate an additional mechanism of action of PARP inhibitors and provide a rationale for combining PARP inhibition with immunotherapies for the treatment of TNBC. SIGNIFICANCE: This work demonstrates cross-talk between PARP inhibition and the tumor microenvironment related to STING/TBK1/IRF3 pathway activation in cancer cells that governs CD8 + T-cell recruitment and antitumor effi cacy. The data provide insight into the mechanism of action of PARP inhibitors in BRCA-associated breast cancer.

Highlights d Unsupervised clustering revealed subtype with EMT and phosphoprotein signatures d Potential therapeutic vulnerabilities included survivin, NSD3, LSD1, and EZH2 d Rb phosphorylation nominated as a biomarker for trials with CDK4/6 inhibitors d Detailed immune landscape analysis highlighted targetable points of immuneregulation

SUMMARY CDK4/6 inhibition is now part of the standard armamentarium for patients with estrogen receptorpositive (ER + ) breast cancer, so that defining mechanisms of resistance is a pressing issue. Here, we identify increased CDK6 expression as a key determinant of acquired resistance after palbociclib treatment in ER + breast cancer cells. CDK6 expression is critical for cellular survival during palbociclib exposure. The increased CDK6 expression observed in resistant cells is dependent on TGF-b pathway suppression via miR-432-5p expression. Exosomal miR-432-5p expression mediates the transfer of the resistance phenotype between neighboring cell populations. Levels of miR-432-5p are higher in primary breast cancers demonstrating CDK4/6 resistance compared to those that are sensitive. These data are Furthermore confirmed in pre-treatment and post-progression biopsies from a parotid cancer patient who had responded to ribociclib, demonstrating the clinical relevance of this mechanism. Finally, the CDK4/6 inhibitor resistance phenotype is reversible in vitro and in vivo by a prolonged drug holiday.

Over the past decade, cancer diagnosis has expanded to include liquid biopsies in addition to tissue biopsies. Liquid biopsies can result in earlier and more accurate diagnosis and more effective monitoring of disease progression than tissue biopsies as samples can be collected frequently. Because of these advantages, liquid biopsies are now used extensively in clinical care. Liquid biopsy samples are analysed for circulating tumour cells (CTCs), cell-free DNA, RNA, proteins and exosomes. CTCs originate from the tumour, play crucial roles in metastasis and carry information on tumour heterogeneity. Multiple single-cell omics approaches allow the characterisation of the molecular makeup of CTCs. It has become evident that CTCs are robust biomarkers for predicting therapy response, clinical development of metastasis and disease progression. This review describes CTC biology, molecular heterogeneity within CTCs and the involvement of EMT in CTC dynamics. In addition, we describe the single-cell multi-omics technologies that have provided insights into the molecular features within therapy-resistant and metastasis-prone CTC populations. Functional studies coupled with integrated multi-omics analyses have the potential to identify therapies that can intervene the functions of CTCs.