IL-4 Mediated Upregulation of AMPK/SIRT1/PGC-1α Signalling Pathway Is Bypassed By Mitochondrial Complex I Downregulation By NAMPT Inhibition in Venetoclax and Ibrutinib Treated CLL Cells

Introduction: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Despite significant improvement in the treatment of CLL, drug resistance is emerging when using the single agents ibrutinib or venetoclax. To achieve greater depth of response, combination treatments are being used to eradicate disease. Altered mitochondrial metabolism is a key factor in CLL survival. In order to gain insights into the underlying biology of a promising drug combination treatment, we investigated the combination of venetoclax and ibrutinib on mitochondrial function as well as the B-cell receptor (BCR), apoptotic and adenosine monophosphate activated protein kinase /silent information regulator 1 / peroxisome proliferator-activated receptor-coactivator-1α (AMPK/SIRT1/PGC-1α) signaling pathways in CLL cells. We also evaluated a proposed mechanism of resistance using interleukin-4 (IL-4) to demonstrate the role of a nicotinamide phosphoribosyltransferase (NAMPT) specific inhibitor, FK866, in order to overcome resistance in vitro.

Methods: Freshly isolated primary B-cells from CLL patients were treated with venetoclax, ibrutinib or their combination in a dose- and -time responsive fashion. CLL cells were also treated with IL-4 and FK866 in the presence or the absence of the combination treatment. Flow cytometry (Novocyte) was used to assess cell viability, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). Mitochondrial respiration rates and specific substrate-dependent respiration of individual complexes of the respiratory chain were measured by respirometry (Orobooros O2k oxygraph) and ATP levels by luminometry (Lmax Luminometer, Molecular Devices). Cellular, mitochondrial, and lysosomal morphology was evaluated by Philips CM10 electron microscope and Olympus BX51 fluorescent microscope. Changes in protein levels of signaling pathways were detected by immunoblotting.

Results: Each single agent venetoclax or ibrutinib reduced mitochondrial respiration profiles in CLL cells in vitro. The combined effect of these drugs on the respiration profiles, ATP, MMP, ROS and cell viability was more profound than with each agent alone. Proteins involved in 1. BCR [Bruton's tyrosine kinase (BTK); serine/threonine-specific protein kinase (AKT); phospholipase Cɣ2 (PLCɣ2) and extracellular signal-regulated kinase (ERK)], 2. Apoptotic B-cell lymphoma 2 (BCL-2); myeloid cell leukemia-1 (MCL-1) and 3. AMPK/SIRT1/PGC-1α signaling in the venetoclax and ibrutinib combination treated samples were significantly reduced when compared to DMSO and each single agent. AMPK/SIRT1/PGC-1α regulated transcription factors responsible for mitochondrial biogenesis [nuclear respiratory factor (NRF1 and NRF2)] and mitochondrial dynamics related proteins [mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1)] were preferentially downregulated by the combination treatment. These effects are seen in the morphological changes, as visualized by transmission electron microscopy demonstrating swelling of mitochondria (venetoclax) and vacuole formation (ibrutinib) in addition to the formation of multi-vesicular bodies in the combination. We also validated the impact of the mitochondria and lysosomes using immunofluorescence. In the presence of IL-4 (a secreted cytokine used to activate the BCR), the effects of the combination were negated by the addition of the NAMPT inhibitor, FK866. FK866 also preferentially decreased mitochondrial respiration rates in the presence of Complex I specific substrates and sustained this inhibition in all FK866 containing conditions regardless of IL-4.

Conclusions: The combined effect of venetoclax and ibrutinib to target mitochondrial metabolism via the AMPK/SIRT1/PGC-1α signaling pathway provides a rationale for this drug combination treatment. The use of IL-4 identifies a potential path of resistance that can be overcome by NAMPT inhibition by directly targeting Complex I of the electron transport chain of the mitochondria.