Abstract 5495: Identification of markers predictive of phosphoramide mustard (PM) cellular sensitivity in lymphoblast cell lines

Abstract

Abstract Cyclophosphamide (Cy) a DNA alkylating agent, is commonly used for treatment of several malignancies such as breast cancer and lymphoma. It is also widely used in conditioning regimens for bone marrow transplantation. Cy is a prodrug requiring activation to 4-hydroxycyclophosphamide by hepatic cytochrome P450 enzymes. 4-hydroxy-Cy non-enzymatically converts to active phosphoramide mustard (PM), which produces inter and intra-strand DNA crosslinks causing DNA breaks and hence the cytotoxicity. Although of great clinical utility the dose limiting side effects of Cy as myelosuppression, nephrotoxicity, cardiotoxicity, neurotoxicity and bladder toxicity posses clinical challenge. Genetic polymorphisms in P450 enzymes especially CYP2B6 as well as phase II drug metabolizing enzymes has been shown to partially explain the inter-patient variation in response in some studies however role of pharmacodynamics players is still not clear. Thus, we designed the present study to identify genes predictive of phosphoramide mustard response. We utilized EBV transformed lymphoblast cell lines (LCLs) that are part of International HapMap project as the model system and treated these with PM to by pass the CYP mediated activation of Cy. Since most of the Cy metabolizing genes (CYPs) are expressed in liver but not as much in LCLs, use of PM instead of Cy in the present study enriched our ability to identify genes of relevance to the pharmacodynamic significance. Briefly we treated LCLs with six different concentrations of PM (100, 50, 20, 10, 1, and 0.1 μM), due to stability issues PM was prepared fresh for each experiment. Cell viability was determined 48 hr post drug treatment by MTT assays. Genome-wide gene expression and genotype data was obtained from publically available source and was evaluated for association with PM cellular sensitivity. Top 100 SNPs (p< 2×10-5) mapped to 10 genes of biological significance including CREBBP, DPP10, HYAL3, XRCC1, SNAPC3, CCDC21, PSIP1, RASSF1, and SALL4 CREBBP (CREB binding protein) is a major transcriptional regulator for both estrogen dependent breast cancer signaling and Lymphotoxin b receptor signaling pathways. CREBBP has an intrinsic histone acetyltransferase activity that acts as a scaffold to stabilize additional protein interactions with the transcription complex. Gene-expression association analysis identified 34 genes significantly associated with PM cytotoxicity including EIF4E2, MMP16, MUC6, NDUFA2, NDUFA9 and SDHB. Identification of SNPs in XRCC1 further confirmed the impact of altered DNA damage repair/response pathway in compromising PM response. Our results identified genetic markers of pharmacodynamic significance to Cy response thus opening up opportunities for clinical validation of these in patients receiving Cy and moving a step closer to personalized medicine. Citation Format: Lata Chauhan, Brooke L. Fridley, Alice Wang, Jatinder Kaur Lamba. Identification of markers predictive of phosphoramide mustard (PM) cellular sensitivity in lymphoblast cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2015-5495