Authors: Elizabeth Katherine Molchan, Kathleen Streeks, Alyssa Ohaegbulam, Mohammed Gbadamosi
Published: 2025-04-22
DOI: 10.1158/1538-7445.am2025-545
Source: Full article
Cyclophosphamide (CTX) is a widely used chemotherapeutic agent in breast cancer (BC) treatment regimens. Despite its frequent use, patient responses vary significantly. While several studies have identified clinically relevant genetic variants associated with CTX metabolism to phosphoramide mustard (PM)—the active cellular anti-tumor metabolite—the factors influencing sensitivity at the cellular level remain poorly characterized. We previously performed a genome-wide association study (GWAS) of lymphoblastoid cell lines identified the SNP rs12408401 as associated with PM resistance. Employing the 15-state chromatin prediction model, ChromHMM, and other in silico tools, we identified an association between this SNP and the disruption of a repressive CTCF binding domain in multiple tissue types, including breast. This leads to an upregulation of several nearby genes regulated by this motif, including RFX5. Analysis of TCGA BC patient data revealed that increased RFX5 expression correlates with reduced disease-free interval in breast cancer patients treated with CTX (HR = 5.32, P = 0.028), but not in other BC patients. RFX5 is a transcription factor that typically regulates the expression of MHC II-related genes. However, RFX5 has been reported to exhibit upregulation in several malignancies and off-target transcriptional activity. Herein, we aim to functionally validate the role of RFX5 in mediating CTX resistance, hypothesizing that decreased RFX5 expression will decrease drug resistance in vitro. To test this hypothesis, we achieved gene knockdown through the administration of silencing RNA (siRNA) targeting RFX5 then assessed sensitivity by treating cells with PM, determining cell viability to quantify IC50. As determined by qPCR, knockdown of RFX5 expression in the cell line MDA-MB-468 resulted in a 91% reduction in RFX5 expression compared to cells treated with scrambled siRNA (p = 0.004). At a PM concentration of 0.4 mM, RFX5 knockdown cells showed significantly reduced viability compared to scramble control cells (33.3 ± 4.5% vs. 49.6 ± 5.4%; p = 0.031). Furthermore, there was a 15% reduction in the IC50 of knockdown cells compared to control cells (0.273 ± 0.016 mM versus 0.346 ± 0.020 mM; P = 0.004). Together, these results provide strong evidence that RFX5 significantly attenuates CTX resistance. This work aims to link SNP genotype, gene expression, and drug sensitivity to improve predictive models of patient response and support targeted therapies enhancing CTX efficacy. Ongoing studies aim to delineate the pathways mediating aberrant RFX5 activity and validate its role in PM sensitivity across additional TNBC cell lines, with a focus on using CRISPRi to investigate the impact of genetic variation on the regulation RFX5 expression and RFX5 target genes.