Supplementary Table 1, Supplemental Figures 1-5 from Protein Kinase D–Dependent Downregulation of Immediate Early Genes through Class IIA Histone Deacetylases in Acute Lymphoblastic Leukemia
posted on 2023-04-03, 19:40authored byGuangyan Sun, Anna Shvab, Guy J. Leclerc, Bin Li, Felipe Beckedorff, Ramin Shiekhattar, Julio C. Barredo
Supplementary Table 1. Antibodies used in the study Figure S1. AICAR-induced downregulation of IEGs mRNA expression in ALL cells. Figure S2. The mechanism of AICAR-induced IEGs downregulation is AMPK-independent. Figure S3. The PKC inhibitor GF109203X suppresses IEGs expression in ALL cells. Figure S4. PKD inhibition downregulates IEGs transcription in ALL cells. Figure S5. The PKD inhibitor CRT synergizes with regorafenib and dexamethasone in ALL cells.
Funding
Batchelor Foundation
Woman's Cancer Association
History
ARTICLE ABSTRACT
Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and adolescents, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. To identify genome-wide metabolic-stress regulated genes, we used RNA-sequencing in ALL cells treated with AICAR, an AMPK activator. RNA-sequencing identified the immediate early genes (IEGs) as a subset of genes downregulated by AICAR. We show that AICAR-induced IEGs downregulation was blocked by an adenosine uptake inhibitor indicating AICAR was responsible for IEGs reprogramming. Using pharmacologic and genetic models we established this mechanism was AMPK-independent. Further investigations using kinase assays, PKD/PKC inhibitors and rescue experiments, demonstrated that AICAR directly inhibited PKD kinase activity and identified PKD as responsible for IEGs downregulation. Mechanistically, PKD inhibition suppressed phosphorylation and nuclear export of class IIa HDACs, which lowered histone H3 acetylation and decreased NFκB(p65) recruitment to IEGs promoters. Finally, PKD inhibition induced apoptosis via DUSP1/DUSP6 downregulation eliciting a DNA damage response. More importantly, ALL patient cells exhibited the same PKD-HDACs-IEGs–mediated mechanism. As proof of principle of the therapeutic potential of targeting PKD, we established the in vivo relevance of our findings using an NSG ALL mouse model. In conclusion, we identified a previously unreported PKD-dependent survival mechanism in response to AICAR-induced cellular stress in ALL through regulation of DUSPs and IEGs' expression.
PKD mediates early transcriptional responses in ALL cells as an adaptive survival mechanism to overcome cellular stress.