Mechanisms Generating free Radicals in CML Stem/Progenitor Cell Populations Causing DNA Damage and Genomic Instability

BCR/ABL kinase is the founding member of a family of oncogenic tyrosine kinases (OTKs) also including TEL/JAK2, TEL/PDGFR, TEL/ABL, and JAK2V617F, which induce myeloproliferative disorders (MPDs). BCR/ABL transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but progenitor-driven disease. In CML-CP, leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) reside in the CD34+CD38- and CD34+CD38+ populations, respectively, whereas in CML-BC, LSCs are also found in the CD34+CD38+ population. In addition, CD34+ CML cells belong to either proliferative or quiescent populations; the latter of which responds poorly to the ABL kinase inhibitors. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations in the kinase domain and additional chromosomal aberrations associated with progression to CML-BC (Oncogene, 2007). Since genomic instability usually results from enhanced DNA damage, we investigated the mechanisms responsible for “spontaneous” DNA damage in cells transformed by BCR/ ABL and other OTKs. Much endogenous DNA damage arises from free radicals such as reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). We showed that CD34+ stem/progenitor CML cells contain higher levels of ROS (superoxide anion = ·O₂⁻, hydrogen peroxide = H₂O₂ and hydroxyl radical = ·OH) and RNS (nitric oxide = NO·) than CD34+ cells from normal donors (CML-BC>CML-CP>Normal). Moreover, ROS levels were elevated in CD34+CD38- and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to the corresponding cells from healthy donor. In addition, both proliferative and quiescent CD34+ CML cell sub-populations contained more ROS than their normal counterparts. Interaction with the stromal cells further elevated ROS levels in BCR/ABL-positive cells. Higher ROS/RNS levels induced more oxidative/nitrative DNA lesions, such as 8-oxoG and DNA double-strand breaks (DSBs), in CML-CP cells resulting in induction of point mutations in BCR/ABL kinase causing imatinib resistance and accumulation of chromosomal aberrations characteristic of CML-BC. In addition, cells transformed by other OTKs also displayed elevated ROS/ RNS and oxidative/nitrative DNA damage, implicating their role in malignant progression of MPDs. Our previous studies showed that elevated levels of oxidative DNA damage in OTK-transformed cells could be diminished by scavenging of ROS with N-acetyl-cysteine and vitamin E, which reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells cultured in vitro and growing in SCID mice (Blood, 2006; Leukemia, 2008). These studies highlighted the importance of identification of the sources of free radicals in CML and other MPDs. We found that elevated levels of ROS in BCR/ABL-transformed cell lines and CD34+ CML cells were generated by three major mechanisms: NADPH oxidase (NOX) complexes containing NOX1 and/or NOX2, complex III of the mitochondrial respiratory chain (MRC), and 5-lipoxygenase (LOX). In addition, inducible nitric oxygen synthase (iNOS) produced RNS in leukemia cells. Using selective inhibitors of NOX, MRC, LOX and iNOS we estimated the contribution of these pathways to accumulation of free radicals causing oxidative/nitrative DNA damage in CML cells. In summary, BCR/ABL kinase-dependent elevation of ROS/RNS depends on several mechanisms, which are now targeted to determine their actual role in genomic instability in CML.