Ancestry-enriched ACKR1 germline variant and its functional impact on normal and breast cancer biology

Atypical chemokine receptor 1 ( ACKR1 ) gene, harbors single nucleotide polymorphisms in its regulatory and coding regions. The regulatory region variant rs2814778 is enriched in African and Arab populations and it confers protection against malarial infection. However, it is responsible for the Duffy-Null (CC) /heterozygous (C/T) phenotype, characterized by a significant reduction in ACKR1 expression in epithelial and non-epithelial cells. ACKR1 functions as a decoy receptor for several chemokines including the breast cancer metastasis-associated CXCL12, to regulate immune/ inflammatory pathways. There is a growing interest to include Duffy genotyping in clinical trial design to account for normal biological differences underlying aggressive breast cancer outcomes in black women. We seek to investigate the potential influence of ACKR1 variant on breast cancer outcomes, with a long-term goal of identifying therapeutic vulnerabilities. We established a model system by generating immortalized breast epithelial cell lines with functional TT (wild type that express ACKR1 -African and European ancestry), heterozygous ( C/T ) and homozygous ( CC ), using breast tissues from the institutional resource of Komen Normal Tissue bank. Cell lines with TT expressed higher ACKR1 mRNA levels relative to C/T or CC in the regulatory region. We hypothesized that reduced ACKR1 expression alters chemokine signaling which may influence intrinsic and extrinsic signaling to activate downstream oncogenic pathways. To test this, we investigated WNT/GSK-3beta signaling and observed that ACKR1 variant influences differential phosphorylation of beta-catenin, facilitating nuclear translocation. We sought to explore further the effect of cytokines including inflammatory IL-6 secreted as a result of ACKR1 variant on downstream oncogenic pathways. We observed increased phosphorylation of STAT3, a surrogate of IL-6 activity in breast epithelial cells of ACKR1 CC and C/T cells compared to TT . Our analysis of the UALCAN database further revealed that reduced ACKR1 expression in breast cancer correlates with progression to brain metastases. Consistent with this, our results demonstrate enrichment of CD271-high populations; indicative of cancer stem-like properties in ACKR1 CC and C/T cells. These findings suggest that reduced ACKR1 expression may promote acquisition of stem-like and metastatic properties. In vivo studies are underway to evaluate the effect of ACKR1 status on distinct tumor characteristics including cancer stem cell properties, increased metastatic potential and response to targeted and conventional chemotherapy. A positive outcome from this study will have a transformative impact, establishing ACKR1 germline variants as determinants of breast cancer biology and highlight the need to integrate ancestry-informed genotypes into clinical trial design and therapeutic decision-making.