ROS-Induced DNA Damage Causing Genomic Instability in CML Stem and/or Progenitor Cells and in Quiescent and/or Proliferating Cells: Role of Mitochondrial Respiratory Chain Complex III.

Abstract 3268

Poster Board III-1

BCR/ABL kinase transforms hematopoietic stem cells to induce chronic myelogenous leukemia (CML). CML in chronic phase (CML-CP) is a leukemia stem cell (LSC)-derived but leukemia progenitor cell (LPC)-driven disease, which is, in most cases, sensitive to ABL tyrosine kinase inhibitors (TKIs) monotherapy. TKIs do not eradicate the leukemia but instead usually render the disease ‘inactive', since the residual quiescent LSCs are intrinsically insensitive to BCR-ABL inhibition and, in a significant cohort of CML patients, LPCs are also refractory or acquire resistance to TKIs due to mutations in BCR/ABL kinase. In the post-imatinib era, these cells may eventually undergo transformation and initiate fatal CML blast crisis (CML-BC). The malignant progression is usually associated with enhanced expression of BCR/ABL and accumulation of additional genetic aberrations, such as TKI-resistant mutations and chromosomal aberrations. In CML-CP, LSCs and 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, LSCs and LPCs usually belong to quiescent (CFSE^max) and proliferative (CFSE^low) populations, respectively. However, the origin of CML-BC clone and the role of BCR/ABL “dosage” are not known. Since genomic instability usually results from DNA damage, we investigated the mechanisms responsible for enhanced DNA damage in CML cells. Much endogenous DNA damage arises from free radicals such as reactive oxygen species (ROS). Here we show that LSCs-enriched CD34+CD38- and quiescent (CFSE^max) CML cells and LPCs-enriched CD34+CD38+ cells contain higher levels of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) than corresponding cells from normal donors (CML-BC>CML-CP>Normal). Interestingly, CFSE^max and CFSE^low CML cells displayed similar elevation of ROS indicating that the presence of BCR/ABL and not the proliferative status enhances ROS. In addition, total cellular ROS and mitochondrial ROS levels were proportional to the expression of BCR/ABL kinase implicating the role of BCR/ABL kinase “dosage”. Higher levels of ROS caused more oxidative DNA lesions, such as 8-oxoG and DNA double-strand breaks (DSBs) in CD34+ and also in CD34+CD38- CML cells than in normal counterparts (CML-BC>CML-CP>Normal). Inhibition of BCR/ABL kinase with imatinib partially reduced ROS and oxidative DNA damage in CD34+ CML-CP cells, implicating BCR/ABL-dependent and -;independent mechanisms. Our previous studies showed that elevated levels of oxidative DNA damage in BCR/ABL-transformed cells were responsible for accumulation of TKI-resistant BCR/ABL mutants and chromosomal aberrations (Blood, 2006; Leukemia, 2008), highlighting the importance of identification of the sources of ROS in CML. Mitochondrial respiratory chain (MRC) is a major site of ATP production via oxidative phosphorylation, which is associated with electron flux through MRC. Some of the electrons may escape and react with molecular oxygen to form ROS. To shut down MRC, cells were depleted of mitochondrial DNA (mtDNA) by long-term exposure to ethidium bromide in the presence of uridine and pyruvate as confirmed by RT-PCR showing the absence/reduction of mtDNA-coded Cox II gene transcript. The absence of functional MRC reduced the level of ROS by 40% and 20% in CD34+ CML-CP cells and normal counterparts, respectively, suggesting that MRC is an important source of ROS in leukemia cells. Using selective inhibitors of various MRC complexes we identified complex III as major producer of ROS in LSCs and LPCs in CML-CP. The role of complex III in CML-BC cells is somehow diminished in concordance with the observation that prolonged exposure of MRC to elevated levels of ROS results in “mitochondrial injury” and reduction of MRC activity in advanced stages of cancer. In summary, we postulate that BCR/ABL kinase generates ROS and oxidative DNA damage in a dose-dependent manner not only in LPCs-enriched CD34+CD38+ and CFSE^low cells, but also in LSCs-enriched CD34+CD38- and CFSE^max cells, and that MRC complex III generates significant amount of ROS in CML-CP cells. Thus, genomic instability causing TKI resistance and progression to CML-BC may originate in LSCs as well as in LPCs.