Abstract 1289: AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia
Authors: Shvab Anna , Sun Guangyan , Li Bin , Beckedorff Felipe , Leclerc Guy J. , Shiekhattar Ramin , Barredo Julio C. 
Published: August 2019

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. We previously uncovered that ALL are vulnerable to metabolic/energy stress and ER-stress via AMP-activated protein kinase (AMPK) activation. In order to identify genome wide metabolic stress and AMPK transcriptionally regulated genes, we used RNA-Seq and compared mRNA profiles in ALL cells treated with the adenosine analog AICAR, an activator of AMPK. RNA-Seq data indicated that high dose AICAR (15 mM/45 min) induced a robust downregulation of a cluster of genes known as the immediate early genes (IEGs), which are critical for cell survival, proliferation and adaptation. AICAR-induced downregulation on IEG expression was dose- and time-dependent, and observed in other cell types (HEK293T, Hela, MEF), indicating this mechanism is conserved in mammalian cells. Using MEF AMPKα2 and AMPKα1/α2 knockout cell lines, we found that these alterations were AMPK-independent. Characterization of AICAR’s mechanism of action identified the protein kinase D1 (PKD1) as responsible for these effects. PKD1 is a Ser/Thr protein kinase involved in many cellular processes important for cancer development and progression, including proliferation, survival, apoptosis, invasion, cell adhesion and angiogenesis. We uncovered that high dose AICAR significantly inhibited PKD1 activation (Ser-910) at the plasma membrane which prevented its nuclear translocation. When PKD1 activity was pharmacologically inhibited by CRT0066101 or downregulated by shRNA, we observed similar IEGs’ downregulation in ALL cells. Conversely, the effect of AICAR on IEGs’ expression was abrogated by PMA, a direct activator of PKD1. In addition, when PKD1 was overexpressed in HEK293T cells, AICAR-induced IEG’s downregulation was partially restored. Using a kinase assay, we found that AICAR, but not ZMP, directly inhibited PKD1 kinase activity. Further, we determined that AICAR suppressed phosphorylation and nuclear export of PKD1-targeted histone deacetylases HDAC4/5, which led to decreased histone H3 acetylation at the IEGs’ promoter region. Finally, ChIP-qPCR indicated that AICAR-induced PKD1 inhibition prevented NF-κB recruitment to IEGs’ promoters. Inhibition of PKD1 activity led to decreased cell proliferation and promoted apoptosis in ALL cells. To confirm the in vivo relevance of our data, single agent and combination experiments using our NSG ALL mouse model are underway. Taken together, we have identified a novel AMPK-independent mechanism leading to AICAR’s inhibition of PKD1-mediated ALL survival. Consequently, co-targeting PDK1 and other pro-survival stress response pathways in ALL cells offers novel strategies to overcome therapeutic resistance.

Citation Format: Anna Shvab, Guangyan Sun, Bin Li, Felipe Beckedorff, Guy J. Leclerc, Ramin Shiekhattar, Julio C. Barredo. AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1289.