Abstract
The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation.
Highlights
We demonstrate that imipramine blue (IB) has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of tyrosine kinase inhibitors (TKI)-insensitive quiescent CD34+ chronic myeloid leukemia (CML) chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro
The IC50 of IB was similar in each of these cell contexts at 1.32 ± 0.25 μM for CD34+ CML-CP cells (n = 3) (Figure 1A), 1.13 ± 0.07 μM for KCL22WT cell line (Figure 1B), and approximately 1.65 μM for imatinib resistant KCL22T315I cells (Supplementary Figure S1A)
Primary CD34+ CML-CP cells were more resistant to apoptosis induction by IB than BCR-ABL1WT+ CML-blast crisis (BC) cell lines, the percentage of Annexin V+ cells increased in a concentration dependent manner in response to IB (Figure 2A–2C), and to a magnitude far greater than can be achieved with TKI
Summary
Double strand breaks and translocations arising from oxidative stress are commonplace with elevated reactive oxygen species (ROS) generated by mitochondrial respiration mediating such DNA damage [1, 2]. Genetic lesions are of particular significance in stem cells. Endosteal niches in which hematopoietic stem cells (HSC) reside provide a hypoxic protective environment conducive to maintaining low ROS and high reconstitution function [3]. Quiescent HSC, despite their relatively high mitochondrial content, exist in a low energy state that restricts DNA damage from replication stress. Proliferating (self-renewing) HSC switch to a high energy state, whereby mitochondrial respiration generates ROS. Mitochondrial ROS can stimulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [4]
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