Acadesine Elicits A Rapid Epigenetic Reprograming Of Immediate Early Genes Through The Protein Kinase D1 Pathway In Acute Lymphoblastic Leukemia Cells Undergoing Energy/Metabolic Stress

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for relapsed/refractory ALL in children and adults remain dismal, highlighting the need for novel targeted therapies capable of overcoming resistance in relapsed/refractory disease. We previously uncovered that ALL cells are vulnerable to metabolic/energy stress and endoplasmic reticulum (ER)-stress via AMP-activated protein kinase (AMPK) activation leading to unfolded protein response (UPR)-mediated apoptosis. In order to identify genome-wide metabolic-stress and AMPK-transcriptionally regulated genes in ALL cells undergoing metabolic/energy stress, we used RNA-Seq and compared mRNA transcript profiles in ALL cells treated with acadesine (adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside or AICAR), known to activate AMPK. RNA-Seq data indicated that acadesine treatment (15 mM/45 min) induced a robust and rapid alteration in gene expression in ALL cells. The most significant acadesine-induced gene signature represented a cluster of genes known as immediate early genes (IEGs), which are fundamental in critical biological pathways for cell survival/proliferation/adaptation. We interpreted these changes as a compensatory pro-survival mechanism in ALL cells undergoing energy/metabolic stress. Among the acadesine-induced downregulated IEGs, we selected DUSP1, JUNB and NFKBIA for further characterization. Downregulation of these IEGs was confirmed using RT-qPCR. We found that the effect of acadesine-induced downregulation on IEGs expression was dose- and time-dependent, and these effects were observed in other cell types (HeLa, HEK293T, mouse embryonic fibroblasts(MEF)), indicating this mechanism of acadesine-induced downregulation of IEGs expression is conserved in mammalian cells. Interestingly, when we used lower doses of acadesine (the half-maximal inhibition concentration for IEGs (IC50)), the IEGs mRNA levels returned to baseline after 3 hours of exposure, suggesting the effect of acadesine on these IEGs was transient at IC50 dose. Using NALM6 AMPKα1 knockdown and MEF AMPKα1/α2 knockout cell lines, we uncovered that high-dose/short-time exposure to acadesine led to changes in IEGs expression that were independent of AMPK. Consistent with these findings, ALL cells co-treated with acadesine plus adenosine kinase inhibitors (ABT702 or 5-Iodotubercidin), which prevent its conversion to ZMP, exhibited the same gene expression signature. Characterization of acadesine's mechanism of action identified protein kinase D1 (PKD1) as responsible for acadesine-induced downregulation of IEGs. PKD1 is a serine/threonine protein kinase involved in many cellular processes important to cancer development and progression, including proliferation, survival, apoptosis, motility, cell adhesion and angiogenesis. Acadesine induced strong inhibition of PKD1 activity which resulted in PKD1 accumulation in the cytoplasm and prevented its nuclear translocation. When ALL cells were treated with protein kinase D (PKD) inhibitors (CRT0066101, GF109203X), we observed a similar rapid, robust and transient downregulation of IEGs, suggesting acadesine interacts with the PKD1 pathway. Conversely, the effect of acadesine on IEGs expression was abrogated by phorbol 12-myristate 13-acetate (PMA), a direct activator of PKD. Further, we determined that acadesine suppresses PKD1-regulated class II Histone deacetylase (HDAC4/5) phosphorylation and nuclear export, which led to decreased histone H3 acetylation levels at the IEG's promoter region. Finally, ChIP-qPCR experiments uncovered that the acadesine/PKD1 axis regulates the recruitment of nuclear factor-κB (NF-κB) to the promoter region of selected IEGs. Consequently, we have identified a novel, AMPK-independent transcription regulation mechanism of acadesine thorugh PKD1 in ALL cells, and co-targeting PDK1 and other pro-survival stress response pathways in ALL cells vulnerable to energy/metabolic stress offers potential novel strategies to overcome therapeutic resistance.