Abstract 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-3β signaling and observed that ACKR1 variant influences differential phosphorylation of β-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. Citation Format: Stephanie Adama, Adedeji Adebayo, Sedat Kacar, Poornima Bhat-Nakshatri, Jiang Guanglong, Cihat Erdogan, Bryan P. Schneider, Kathy D. Miller, Harikrishna Nakshatri. Ancestry-enriched ACKR1 germline variant and its functional impact on normal and breast cancer biology [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 6287.

Abstract PIK3CA is the second most mutated and/or amplified gene in breast cancer after p53. The PIK3CA-specific inhibitor Alpelisib is an FDA approved treatment for breast cancer. Toxicity and rapid development of resistance limit its clinical utility. Mutations in PIK3CA are found in many normal organs including the breast suggesting that additional genomic aberrations are needed for mutant PIK3CA to initiate breast cancer, and those aberrations can be exploited for therapy. Consistent with this possibility, our previous studies have demonstrated that immortalized breast epithelial cells derived from BRCA1/2 mutation carriers are susceptible to transformation by the PIK3CA mutant, whereas transformation of immortalized breast epithelial cells from healthy donors required combinations of mutant PIK3CA and SV40-T/t antigens. These results suggest that transformation of breast epithelial cells by PIK3CA mutants requires additional genomic aberrations like those induced by SV40-T/t antigens or BRCA1/2 mutations. To decipher genomic aberrations required for PIK3CA mutant driven breast epithelial transformation, we used model system of immortalized breast luminal epithelial cell lines from healthy donors. We show that mutations that activate MEK/ERK pathway such as NF-1 mutation in parallel with PIK3CA mutations initiate ductal carcinoma- in situ (DCIS)-like breast tumors with accompanying reduction in BRCA2 protein, whereas mutant PIK3CA plus SV40-T/t antigens generate invasive ductal adenocarcinoma (IDC). DCIS and IDC cells differed significantly in the expression of extracellular matrix components including reduced expression of LAMC2 and COL17A1 and upregulation of drivers of aneuploidy in IDC cells. Consistent with the possibility of mutant PIK3CA and MEK/ERK pathway collaboration in breast cancer initiation, Estrogen Receptor-positive/PIK3CA mutation-positive breast tumors contained higher levels of activated ERK compared to Estrogen Receptor-positive/PIK3CA-wild type breast cancers. Significance: To our knowledge, this is the first study to generate DCIS like lesions from breast epithelial cells from healthy women using combinations of breast cancer-enriched genomic aberrations and without the use of viral proteins such as SV40-T/t antigens or HPV E6. In this process, we have developed an isogenic DCIS and IDC model to study breast cancer progression. These results also suggest that mutant PIK3CA is a weaker oncogene and the effective therapy for mutant PIK3CA driven breast cancers needs to include inhibitors of PIK3CA as well as drugs that target genomic aberrations that cooperate with mutant PIK3CA. ​ Citation Format: Snehal B. Bhandare, Ruizhong Wang, Poornima Bhat-Nakshatri, Stephanie Adama, Sarah R. Spivak, Farzaneh Behzadnia, Cihat Erdogan, Hongyu Gao, Yunlong Liu, Lylah H. Hutson, Xin Dauterman, George Sandusky, Harikrishna Nakshatri. Genomic determinants of PIK3CA mutation driven breast cancer initiation [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 3006.

Abstract Pregnancy, lactation, and involution have opposing effects on breast cancer incidence and tumor aggressiveness. While breast feeding and subsequent involution protects against breast cancers, pregnancy-associated breast cancers (breast cancers diagnosed during pregnancy and within 2 years postpartum) are associated with poor outcome. Clear understanding of these opposing effects of reproductive cycle needs single cell resolution of breasts during pregnancy, lactation and involution. We recently reported a reference single nucleus chromatin accessibility and transcriptome atlas of breast tissues of women of diverse genetic ancestry and reported 10 major cell types in the breasts. These include three epithelial cell types [luminal hormone sensing (LHS), luminal adaptive secretory precursors (LASP), and basal-myoepithelial cells (BM)], two adipocyte subtypes, two endothelial cell subtypes, T cells, macrophages, and fibroblasts. In this study, we generated single nucleus atlas of breast tissues of healthy women collected during pregnancy, lactation, and involution. Pregnancy, lactation, and involution are associated with dramatic changes in cell type proportions and cell state shifts within a cell type. LASP cell numbers increased from 24% in the normal reference breasts to 71% during pregnancy, 63% during lactation but returned to 25% during involution. There is also a dramatic shift in LASP cell states during pregnancy, lactation and involution. While the LASP cells in the normal reference breasts and breasts during pregnancy were in both LASP-basal-luminal (LASP-BL) and LASP-alveolar (LASP-AP) states, LASP-AP cells were dominant in lactating and involuting breasts. Increase in LASP cells during pregnancy and lactation is at the expense of BM cells, which reduced from 27% in the normal reference breasts to 7-9% during pregnancy and lactation. While T cell proportion was similar between normal reference and pregnant breasts (2-3%), it increased to 7-8% during lactation and involution, which a recent study has suggested to be responsible for breast feeding-associated protection against breast cancer. Increase in T cells during lactation and involution is likely due to expansion of tissue resident T (TRM) cells as T cells of lactating and involuting breasts expressed higher levels of TRM cell markers CD69 and CXCR6. We present pregnancy, lactation and involution-associated chromatin accessibility and transcriptome changes at individual gene and cell type levels. Several biomarkers associated with these changes have been identified, which will be a useful resource to mechanistically evaluate evolution of breasts during reproductive history and breast cancer development. Citation Format: Poornima Bhat-Nakshatri, Cihat Erdogan, Hongyu Gao, Yunlong Liu, Rana German, Michele L. Cote, Harikrishna Nakshatri. Pregnancy, lactation, and involution-induced remodeling and cell state changes in breast tissues of healthy women [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 6326.

1097 Background: Homologous Recombination (HR) and Replication Stress (RS) resolution mechanisms help to maintain genomic stability. TONSL, MMS22L, RAD51, BRCA1/2 are the few proteins that cooperatively coordinate HR and RS resolution. Therefore, genes in these pathways are expected to function as tumor suppressors. However, only BRCA 1/2 function as tumor suppressors as their germline mutations increase cancer susceptibility. TONSL and RAD51 are frequently overexpressed in tumors. Here, we investigated the relationship between TONSL-RAD51 axis in breast cancer (BC) outcome and response to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET). Methods: 10,980 ER+ BC samples tested by NGS (592, NextSeq; WES/WTS, NovaSeq, Caris Life Sciences). Dual expression group TONSL/RAD51 -high (H), TONSL -H/ RAD51 -low (L), TONSL -L/ RAD51 -H, and TONSL / RAD51 -L were classified by RNA expression above or below median. HRD score and TONSL /HRD-H/L survival was analyzed using METABRIC (ER+ BC, n = 1904) dataset. TONSL-RAD51 role in sensitivity to CDK4/6i was determined using TONSL -amplified ER+ MCF-7 cells. Real-world median overall survival (mOS) of TONSL/RAD51 groups was derived from insurance claims and calculated from biopsy, start of ET or CDK4/6i to last contact using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U with multiple comparison adjustments (q<0.05). Results: TONSL -H had higher HRD score (33.7 vs 21.1, p<0.01) compared to TONSL -L (n=952 each). TONSL /HRD-H (n=573) had worse mOS (109 m vs 138 m, p<0.05) compared to TONSL /HRD-L (n=630). TONSL -H had higher frequency of LOH (31.6% vs 19.8%), BRCA2 (5.1% vs 2.9%) and BRCA1 (1.5% vs 0.8%) mutation, all q<0.05. TONSL/RAD51 -H had worse mOS compared to TONSL -H/ RAD51 -L, TONSL -L/ RAD51 -H or TONSL / RAD51 -L (Table). TONSL -H/ RAD51 -L had worse survival with ET compared to other groups. With ET+CDK4/6i, TONSL/RAD51 -H and TONSL- H /RAD51 -L had similar but worse mOS compared to TONSL -L/ RAD51 -H and TONSL / RAD51 -L (Table). TONSL knockdown in CDK4/6i resistant MCF-7 cells created TONSL -L/ RAD51 -H phenotype and cells regained the sensitivity to CDK4/6i. Conclusions: These findings suggest that TONSL -H independent of RAD51 expression is associated with poor response to ET or ET+CDK4/6i. Therapeutic targeting of TONSL to create TONSL-L/RAD51-H status may improve response to ET+CDK4/6i. Moreover, as the activity of TONSL is regulated by various protein complexes, destabilizers of TONSL-protein complexes could potentially help in sensitizing ER+ BC to ET or CDK4/6i. TONSL/RAD51 -HOS in months TONSL -H/ RAD51 -LOS in months TONSL-L/RAD51 -HOS in months TONSL/RAD51 -LOS in months p-value Overall 35 m (n=4176) 41.4 m (n=1473) 37.9 m (n=1476) 44.3 m (n=4173) <0.01 ET 63.4 m (n=1361) 58 m (n=461) 65.2 m (n=508) 76.1 m (n=1430) <0.01 ET+CDK4/6i 73.8 m (n=2261) 73.8 m (n=866) 84.6 m (n=799) 80.7 m (n=2291) <0.01

International efforts focused on discovery of biological targets in inflammatory breast cancer (IBC) have suggested that aberrant inter-cellular relationship instead of genomic aberrations is the key driver of IBC. To identify such IBC-specific aberrations, we generated single nucleus chromatin accessibility and transcriptome atlas of IBCs (9,628 nuclei from three donors) and compared IBC atlas with a similar atlas of the healthy breast (81,735 nuclei from 92 donors). We recently reported single nucleus atlas of breast tissues of healthy women of diverse genetic ancestry (Nature Medicine in press). In that report, we described markers that identify three major epithelial cell subtypes [Basal-myoepithelial (BM), luminal adaptive secretory precursor (LASP), luminal hormone sensing (LHS)], two endothelial cell subtypes, two adipocyte subtypes, fibroblasts, macrophages, and T cells of the healthy breast. We also showed that LHS cells are the likely cells-of-origin of Luminal A, Luminal B and HER2+ breast cancers. Unlike these breast cancer subtypes, IBC does not appear to have a cell-of-origin, as epithelial cells in IBCs shared gene expression pattern across BM, LASP and LHS cells. Similar results were obtained when comparison was restricted to ancestry-specific healthy breast atlas. However, individual gene level expression differences affecting specific signaling pathways were observed in epithelial cells of IBCs. LASP, and LHS cells of IBCs overexpressed GPR137C, a positive regulator of mTORC1 signaling, and HS6ST3, an enzyme required for heparan sulfate synthesis. LASP and LHS cells of IBCs displayed activation of mitotic and matrix metalloproteinase signaling, respectively. Endothelial cells of IBCs, which showed significant gene expression differences compared to endothelial cells of the healthy breast, displayed enhanced integrin cell surface interaction but loss of cell junction organization. Most critically, BM, LASP, LHS, and endothelial cells of IBCs compared to their counterparts in the healthy breast showed downregulation of TEX14, an inter-cellular bridge forming factor in germ cells and a regulator of mitosis. Downregulation of TEX14 expression in these cells was accompanied with changes in the chromatin accessibility patterns of this gene. These results reinforce the notion that defective inter-cellular communication is a hallmark of IBC and TEX14 expression levels may serve as a biomarker of this defect. We suggest that greater attention to vascular biology and vascular-epithelial cell communication has to be given for better understanding of IBC biology and therapeutic targeting. Citation Format: Harikrishna Nakshatri, Poornima Bhat-Nakshatri, Cihat Erdogan, Hongyu Gao, Yunlong Liu. Single nucleus chromatin accessibility and transcriptome analyses reveal aberrant inter-cellular communication in inflammatory 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 P3-05-23.

Table S13: Enrichment of binding sites for NF-kB and p53 in chromatin regions that become accessible or closed, respectively, upon TONSL overexpression. Genes names and chromatin positions are indicated.

Table S2: Gene expression differences between mature luminal and luminal progenitor cells of the breast. Data are from 15 samples, five each from women of European, African and Latina ancestry.

Contains additional materials and methods and supplementary figures

Table S8: Multivariable analysis for overall (N=469) and progression-free (N=453) survival NOT including HER2. Overall and Progression-Free survival data of patients with ER+ breast cancer and treated with endocrine therapy are also shown.

Table S1: Gene expression differences between primary and hTERT-immortalized breast epithelial cell lines.

Table S15: List of genes differentially expressed upon TONSL overexpression in primary cells with genes differentially expressed in TMD-436 upon TONSL knockdown. Fold changes in gene expression are indicated.

Table S5: Description of the patients and characteristics of their tumors (n=597)

Table S11: The effects of TONSL overexpression on proliferation signature of breast tumors.

Table S9: Results of ATAC-seq of primary and TONSL-immortalized cells.

Table S14: Genes differentially expressed in TMD-436pLKO and TMD-436shTONSL cells. Fold changes in gene expression are indicated.

Table S10: Results of RNA-seq analysis of primary and TONSL-immortalized cells. Fold differences in expression are shown.

Table S12: List of interactors and antagonists of BRCA1-BRAD1 complex. Fold changes in gene expression are indicated.

Table S4: Breast cancers with TONSL amplification selectively overexpress genes associated with E2F targets, G2/M checkpoint, mitotic spindle and mTORC1 pathways.

Table S3: DepMap analysis reveals gene essentiality for survival of select genes upregulated in immortalized cells compared to primary cells.

Table S7: Univariable analysis of other tumor markers for overall and progression-free survival