Abstract 3781: The ribonucleoside analog, 8-chloro-adenosine, induces autophagy in primary chronic lymphocytic leukemia cells in vitro and in vivo
Authors: Stellrecht Christine M. , Balakrishnan Kumudha , Dennison Jennifer B. , Shentu Shujun , Ayres Mary , Wierda William G. , Gandhi Varsha 
Published: June 2012

Abstract
The nucleoside analog, 8-chloro-adenosine (8-Cl-Ado) is in a phase I clinical trial for chronic lymphocytic leukemia (CLL). In contrast to deoxyribose or arabinose containing analogs that are currently established for cancer therapeutics, this unique nucleoside analog possesses a ribose sugar and thus is RNA directed. Previously, we demonstrated that in primary CLL cells, 8-Cl-Ado accumulates as a triphosphate reaching a plateau of ∼400 μM by 6 h of treatment with 10 μM 8-Cl-Ado [Blood 105:4455, 2005]. This accumulation was coupled with a parallel decrease in the level of the endogenous ATP pool by ∼60%. These changes in nucleoside levels elicit an inhibition of RNA synthesis by up to 70%. Because 8-Cl-Ado treatment leads cells to become bioenergetically compromised, we further investigated the effect of this phenomenon on primary CLL cells. Depletion of the cellular ATP pool is often associated with the activation of AMP-activated protein kinase (AMPK) through its phosphorylation at Thr172. Once activated, AMPK then stimulates various energy regulating signaling cascades, most notable of which is the induction of autophagy. Because 8-Cl-Ado readily depletes the ATP pool, we tested if AMPK activity was affected. Using immunoblot analysis, we demonstrated that while total AMPK protein levels were not perturbed by 8-Cl-Ado treatment, the phosphorylation of Thr172 AMPK was induced in a dose dependent manner (3-10 μM) and was associated with an increase in the phosphorylation of an AMPK target, acetyl-CoA carboxylase. To determine if 8-Cl-Ado induces autophagy in primary CLL cells, we examined the cells after 8-Cl-Ado treatment for changes in microtubule-associated protein 1 light chain 3B (LC3B) and showed there was an increase conversion to its lipidated form, which is a marker for autophagosome formation. Also consistent with the induction of autophagy, 8-Cl-Ado promoted the formation autophagolysosomes as shown by acridine orange staining of the CLL cells. To further extend our observations, we tested induction of autophagy in CLL lymphocytes obtained from patients accrued on ongoing 8-Cl-Ado clinical trial. Patients received a 1-hr infusion of 8-Cl-Ado at escalating doses (45, 67.5, or 100 mg/m2/day × 5 day) and peripheral blood samples were obtained both pre- and post infusion. Induction of LC3B lipidation was seen as early as 3 hr post infusion in circulating CLL cells. Additionally, acridine orange staining was increased in these lymphocytes. Finally, p62/SQSTM1 levels fluctuated during and between infusions with the levels decreasing during infusion and rebounded prior to infusion on consecutive days which would be indicative of autophagic flux. In conclusion, our data demonstrate that 8-Cl-Ado treatment induces autophagy in CLL lymphocytes during in vitro incubations as well as in vivo during clinical trial.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2011-3781