Abstract 994: Role of tumor hypoxia in mediating cellular resistance to Gemcitabine and Cytarabine.

Gemcitabine and Cytarabine (Ara-C) are commonly used anticancer nucleoside analogs. These cytotoxic agents, upon cellular uptake by nucleoside transporters such as SLC29A1, are activated by multiple phosphorylation steps, with deoxycytidine kinase (dCK) catalyzing first rate-limiting step. Previous studies have shown development of resistance to anticancer agents in hypoxia mainly due to activation of signaling pathways that regulate processes like cell proliferation, angiogenesis and apoptosis. However, hypoxic conditions can influence intracellular levels of gemcitabine/Ara-C by altering expression levels of genes involved in their influx and activation/inactivation. Additionally, hypoxic environment can alter miRNA levels, thereby indirectly influencing expression levels of gemcitabine/Ara-C pathway genes. Thus, the aim of the study is to characterize the expression patterns of gemcitabine/Ara-C pathway genes as well as miRNAs targeting these genes, under hypoxic conditions, in order to understand the molecular mechanisms underlying role of hypoxia in altering cellular sensitivity to nucleoside analogs. We evaluated lymphoblast cell lines (LCLs) that are part of International HapMap project for impact of hypoxia on gemcitabine/Ara-C cellular sensitivity and determined expression levels of 8 genes in gemcitabine/Ara-C metabolic pathway. We observed higher IC 50 values for gemcitabine (52.71 ± 9.97nM vs 19.12 ± 2.11nM) and Ara-C (14.15 ± 7.11μM vs 4.83 ± 0.8μM) in hypoxic conditions as compared to normoxia. The mRNA expression levels of gemcitabine/Ara-C pathway genes showed nearly 1.5-fold decrease in dCK levels and 3.9-fold increase in CDA levels after 48h in hypoxia as compared to normoxia. The pharmacodynamic targets RRM1 and CTPS were found to be 3.3-fold and 10-fold lower, respectively. Interestingly, there was 1.3-fold increase in expression of SLC29A1 following 48h in hypoxia. We observed no differences in expression of NT5C2, NT5C3 and RRM2 genes under hypoxic conditions. We are also currently evaluating the miRNAs with binding sites in gemcitabine/Ara-C metabolic pathway genes for association with mRNA levels of these genes, as well as with cellular cytotoxicity to gemcitabine/Ara-C in normoxia and hypoxia. In conclusion, our study demonstrates that cellular resistance to gemcitabine/Ara-C under hypoxic conditions could be partly explained by alteration in expression of disposition genes of these nucleoside analogs. Future studies would involve understanding molecular mechanisms underlying hypoxia-mediated resistance to these agents by evaluating mRNA and miRNA expression levels, and their relationship with cellular sensitivity. Additionally, we plan to determine the effect of genetic polymorphisms on hypoxia-mediated cellular resistance in gemcitabine and Ara-C. Funding:This work was supported by NIH R01-CA132946 (Lamba) Citation Format: Neha S. Bhise, Jatinder K. Lamba, Department of Experimental and Clinical Pharmacology, PUMA-Institute of Personalized Medicine. Role of tumor hypoxia in mediating cellular resistance to Gemcitabine and Cytarabine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2013-994