Abstract
Abstract 1211
Background:
BCR-ABL1 –positive chronic myeloid leukemia in chronic phase (CML-CP) is a leukemia stem cell (LSC)-derived but leukemia progenitor cell (LPC)-driven disease, which may eventually progress to fatal CML blast phase (CML-BP). In CML-CP, LSCs and LPCs reside in the CD34+CD38- and CD34+CD38+ populations, respectively. In addition, majority of LSCs and LPCs belong to quiescent (CFSEmax) and proliferative (CFSElow) populations, respectively. Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib do not eradicate the leukemia but instead render the disease ‘dormant’. Residual quiescent LSCs are intrinsically insensitive to TKIs and, in a significant cohort of CML patients, LPCs acquire resistance to TKIs. In the TKI era, these cells may eventually initiate disease relapse and progression to CML-BP, which is associated with genomic instability manifested by accumulation of TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations. Previously we showed that BCR-ABL1 kinase stimulates reactive oxygen species (ROS)-dependent oxidative DNA damage resulting in genomic instability (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). These studies highlighted the importance of identification of cellular lineage origin and mechanisms responsible for generation of ROS-mediated oxidative DNA damage in CML.
Result:
Here we show that LSC-enriched CD34+CD38- and quiescent (CFSEmax) CML-CP cells and LPC-enriched CD34+CD38+ and proliferating (CFSElow) CML-CP cells contain higher levels of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and oxidative DNA lesions (8-oxoG and DNA double-strand breaks) than corresponding cells from healthy donors. Non-mutated and TKI-resistant BCR-ABL1 kinase mutants (Y253F, T315I, H396P) stimulated ROS-induced oxidative DNA damage in a BCR-ABL1 dosage-dependent manner. Inhibition of BCR-ABL1 kinase with imatinib only partially reduced ROS and oxidative DNA damage in CD34+ CML-CP cells, implicating kinase-dependent and –independent mechanisms.
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. We detected that CD34+ CML-CP cells displayed lower mitochondrial potential than normal counterparts, which is indicative of enhanced ROS production. To determine the role of MRC in ROS-induced oxidative DNA damage, cells were depleted of mitochondrial DNA (mtDNA) by ethidium bromide, as confirmed by RT-PCR showing the absence/reduction of mtDNA-coded Cox II gene transcript (Rho0 cells). The absence of functional MRC reduced ROS and oxidative DNA damage in CD34+ CML-CP Rho0 cells and 32Dcl3 Rho0 cells transformed by non-mutated and TKI-resistant BCR-ABL1 kinase mutants, but not in normal counterparts, implicating a specific role of MRC in genomic instability in leukemia cells. In concordance, BCR-ABL1 –positive 32Dcl3 Rho0 cells accumulated fewer TKI-resistant BCR-ABL1 kinase mutants than cells with functional MRC. Using selective inhibitors of various MRC complexes we identified complex III as major producer of ROS and oxidative DNA damage in CD34+CD38- and quiescent LSCs and in CD34+CD38+ and proliferating LPCs in CML-CP. Moreover, BCR-ABL1 –positive cells in which complex III was inactive due to a single base substitution within the cytochrome b gene displayed diminished capability to generate ROS. In contrast, ROS was not affected in cells lacking complex I due to a mutation in the ND6 gene. In addition to BCR-ABL1 –positive CML-CP complex III also appeared to play a major role in generation of ROS in FLT3(ITD)-positive acute myeloid leukemia cells and in JAK2(V617F)-positive polycythemia vera cells.
Conclusion:
In summary, we postulate that enhanced production of ROS by MRC complex III induces genomic instability in LSC-enriched CD34+CD38- and quiescent cells, and also in LPC-enriched CD34+CD38+ and proliferating cells. Thus, genomic instability causing TKI resistance, disease relapse and progression to CML-BP may originate in LSCs as well as in LPCs.
Disclosures:
No relevant conflicts of interest to declare.
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