Abstract 673: Vulnerability of LKB1 deficient NSCLC to C8-modified adenosine analogs is associated with diminished autophagy induction
Authors: Stellrecht Christine M. , Chen Lisa S. , Ayres Mary L. , Heymach John V. , Gandhi Varsha 
Published: August 2015

The C8-modified adenosine analogs, 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-NH2-Ado) are cytotoxic to a number of liquid and solid tumor cells. Compared with nucleoside analogs established for cancer therapeutics, these analogs are unique as they possess a ribose sugar. These distinctive analogs deplete cellular ATP and are RNA directed and 8-Cl-Ado, is currently in a phase I clinical trial for hematological malignancies. Previously, using a screen of non-small cell lung cancer (NSCLC) cell lines for growth sensitivity against a panel of therapeutic agents, we established that LKB1 mutant NSCLC cells were highly resistant to standard chemotherapy agents but showed high sensitivity to 8-Cl-Ado. This sensitivity was diminished in cells made LKB1-proficient [Cancer Res 74s19:5480, 2014].

LKB1 promotes the maintenance of the cellular energy balance by enhancing the activation of AMP activating protein kinase (AMPK) under conditions of reduced ATP levels. Consequently, LKB1 deficient tumors lack proper energy regulation, thus are vulnerable to ATP depletion. To examine the tumoricidal activity of 8-Cl-Ado in NSCLC, we assessed isogenic A549 LKB1 proficient and deficient cell lines for 8-Cl-Ado-induced death by flow cytometry. 8-Cl-Ado was tumoricidal to these cells with greater sensitivity in the LKB1 deficient cells, (38 versus 23% annexin V/propidium iodide positivity with 20 μM 8-Cl-Ado for 4d, p = 0.001). HPLC analysis indicated that the endogenous ATP levels are lower in LKB1 deficient cell lines than LKB1 proficient cells (2800 versus 3400 μM, p = 0.011, respectively in the H460 cells and 2556 versus 2812 μM, p = 0.38, respectively in the A549 cells). This was further perturbed by 8-Cl-Ado treatment as the ATP levels were depleted to a greater extent in the deficient cells compared with the proficient cells (60 versus 40% reduction, p = 0.002, respectively in the H460 cells and 67 versus 52% reduction, p = 0.023, respectively in the A549 cells). In concert, 8-Cl-Ado-induced AMPK phosphorylation was detected in LKB1 proficient cells. Due to AMPK's ability to promote autophagy for energy restoration and the role of autophagy in chemoresistance, we examined the NSCLC cells for 8-Cl-Ado-induced changes in autophagy and demonstrated significantly greater LC3B-lipidation and acridine orange staining (30% higher, p = 0.014) in the LKB1 proficient cells. We also initiated studies with 8-NH2-Ado and demonstrated there was higher metabolism of the analog to its cytotoxic form in the NSCLC cell lines as compared with 8-Cl-Ado. This was associated with enhanced tumoricidal activity in the LKB1 deficient cells with cell death occurring earlier after 1 d of treatment. Our results indicate that LKB1 mutant NSCLC cells are vulnerable to energy altering effects of C8-modified adenosine analogs and that the protective role LKB1 plays during treatment of these cells is due in part to greater induction of autophagy.

Citation Format: Christine M. Stellrecht, Lisa S. Chen, Mary L. Ayres, John V. Heymach, Varsha Gandhi. Vulnerability of LKB1 deficient NSCLC to C8-modified adenosine analogs is associated with diminished autophagy induction. [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 673. doi:10.1158/1538-7445.AM2015-673