ANP32E drives vulnerabiliy to ATR inhibitors in Triple Negative Breast Cancer by accumulation of R-loops [RNA-Seq]

Triple Negative Breast Cancer (TNBC) is often characterized by MYC oncogene overactivation, causing dysregulation of gene expression and uncontrolled proliferation. We found that the expression of ANP32E chaperone protein is increased in dependency of MYC in human mammary epithelial cells, with associated induction of genome instability. TCGA data confirmed that ANP32E is overexpressed in TNBCs with respect to the other BC subtypes. TNBC patients show higher frequency of genomic mutations, correlating with higher ANP32E expression. Investigation of the DNA damage response (DDR) pathways showed an upregulation of several involved genes in correlation with ANP32E overexpression. We demonstrated that inhibiting the DDR master regulator ATR triggers vulnerability of TNBC overexpressing ANP32E in vivo and leads to Double-Strand Breaks and micronuclei accumulation in vitro. We hypothesized the involvement of R-loops (DNA:RNA hybrids) in DDR sensitization, due to their accumulation after ANP32E overexpression and the generation of R-loop dependent Transcription Replication Conflicts. To clarify the underlying mechanism, we tested several DDR players, showing that ANP32E overexpressing cells have: i) sustained stimulation of DDR measured as phRPA32, phChk1, γH2A.X and 53BP1 activation; ii) enhanced recruitment of DDR players to specific chromatin loci; iii) R-loops accumulation at ANP32E chromatin bound sites; ii) elongating RNApolII stalled at R-loops sites, resulting in reduced nascent RNA. With the advantage of Next Generation Sequencing technologies, we mapped R-loops genome wide and observed an accumulation of longer, thus more stable, R-loops in dependency to ANP32E overexpression. Those sites also showed an increased phosphorylation of RPA32, a surrogate marker for Transcription Replication Conflicts. The consequent dependency of cancers with such biological condition from a constant activation of DDR puts the bases for a successful therapy based on ATR inhibitor drugs, which is also empowered thanks to the synergic effect with anti-proliferative molecules such as Navitoclax. In summary, dissection of the molecular mechanism linking ANP32E overexpression to the dependency on DDR specific players and fragile sites will help to rationally design patient-specific therapies and identify novel biomarkers for TNBC diagnosis.