Introduction: Although increased phosphoethanolamine (PE) has been observed consistently in tumors, understanding the role of PE in cancer is relatively unexplored. PE is synthesized from ethanolamine either through the dual choline/ethanolamine kinase activity of choline kinase (ChK)-α or β or the ethanolamine kinase activity of ethanolamine kinase (EtnK)-1 or 2. We have previously shown that EtnK-1 has a major role in maintaining PE levels in vivo in breast cancer cells (1). Here we have further investigated the role of ethanolamine kinase-1 (EtnK-1) and its contribution to PE levels in pancreatic cancers. We have examined the effect of small interfering RNA (siRNA) downregulation of ChK and EtnK on cell viability as a potential therapeutic strategy in these pancreatic cancer cells. HPNE-hTERT (HPNE), Panc-1, Pa02c, Pa04c pancreatic cell lines were cultured in their culture medium supplemented with 50µM ethanolamine. While HPNE is an immortalized pancreatic cell line, Panc-1 is a primary adenocarcinoma cell line. Pa02c and Pa04c were derived from liver and lung metastasis of adenocarcinomas, respectively. High-resolution ³¹P MR spectra from water soluble cell extracts of the pancreatic cell lines were obtained. Water soluble fractions were collected, lyophilized and dissolved in 0.6 ml deuterated water containing phenylphosphonic acid (PPA) that served as a concentration standard as well as a chemical shift reference. ³¹P MR spectra were acquired on a Bruker 11.7T MR spectrometer using composite pulse proton decoupling. qPCR was carried out to determine the basal EtnK1 message level in these pancreatic cells lines. ΔCt cycles of EtnK1 were determined relative to 18S rRNA as an internal control. For assaying cell viability, 4000 cells per well were plated in a 96 well plate and transfected with 50nM siRNA and viability assessed 96h post transfection using the Cell Counting Kit-8 according to the manufacturer’s protocol. While 50nM siRNA was used in all individual siRNA treatment, for combination siRNA treatment 50nM of each specific siRNA was used. All siRNAs were custom designed using the Thermo Scientific siRNA design center. Representative ³¹P MR spectra of the PC and PE regions obtained from pancreatic cancer cells cultured in ethanolamine containing medium are shown in Figure 1A. Quantitative PC and PE levels obtained from the spectra are presented in Figure 1B. Variable levels of PE were observed in these pancreatic cell lines. Pa04c showed the highest level of PE, followed by Panc-1 and HPNE. Pa02c did not show any detectable levels of PE. ΔCt cycles of EtnK1 obtained from pancreatic cell lines are shown in Figure 1C. While Pa04c cells took the least number of cycles to form a product showing an abundance of EtnK1 message, Pa02c took most number of cycles to form a PCR product. Figure 2 shows cell viability data following treatment with ChK and EtnK siRNA in pancreatic cell lines. Pa04c and Panc-1 showed a significant reduction in cell viability compared to untreated condition (p<0.05) on ChK-α or EtnK-1 siRNA treatment. Pa04c, which showed the highest levels of PE, also showed the most reduction in cell viability. Interestingly, HPNE, which is a non-cancerous pancreatic cell line also showed dependency on PE in their growth, together with a significant reduction in cell viability following EtnK-1 down-regulation (p<0.05). EtnK-1 siRNA treatment in Pa02c did not show any reduction in cell viability compared to D-FECT that corroborates the low EtnK-1 activity in these cells. EtnK-2 siRNA treatment did not show any significant effect on cell viability in these pancreatic cell lines. Combined treatment with ChK-α+EtnK-1 siRNA carried out in Panc-1 and Pa04c was not more effective than treatment with the individual siRNA alone, suggesting that both siRNA act through the same pathway in decreasing cell viability. The reduction in cell viability caused by treatment with EtnK-1 siRNA indicates its therapeutic potential that warrants further investigation. Further studies are underway to investigate PE levels in pancreatic tumors in vivo.