Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII–generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor–resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)–positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

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445 For decades, chronic myeloid leukemia (CML) has served not only as a paradigm for understanding the evolution and multi-step process of carcinogenesis but also for studying cancer stem and progenitor cells responsible for the initiation and/or maintenance of the disease. CML is initiated by BCR-ABL1 tyrosine kinase transformation of hematopoietic stem cells into leukemia stem cells (LSCs) to induce CML-chronic phase (CML-CP). The deregulated growth of LSC-derived leukemia progenitor cells (LPCs) leads to manifestation of the disease. It is unclear if LSCs and/or LPCs are able to acquire additional genetic changes that confer resistance to tyrosine kinase inhibitors (TKIs) and induce more aggressive CML blast phase (CML-BP). In addition, the mechanisms and consequences of genomic instability may differ substantially among these cells. For example, the effects of genetic aberrations acquired in quiescent LSCs may be dormant, but if the aberrations induce proliferation or appear in LSCs that are already cycling, they may generate TKI-resistant and/or more malignant clones. Alternatively, genomic instability in LPCs must be accompanied by the acquisition of LSC-like properties to prevent mutations from disappearing before they undergo terminal maturation. Previously, we reported that BCR-ABL1–transformed cell lines accumulate reactive oxygen species (ROS)-induced oxidative DNA damage [8-oxoguanine (8oxoG), double strand breaks (DSBs)] resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). To determine which populations of CML-CP cells, LSCs and/or LPCs, accumulate genomic instability we employed the SCLtTA/BCR-ABL1 tetracycline-inducible (tet-off) transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood. BCR-ABL1 mRNA expression was higher in the Lin−c-Kit+Sca1+ murine leukemia stem cell–enriched population (muLSCs) than in the Lin−c-Kit+Sca1− murine leukemia progenitor cell–enriched population (muLPCs), thus reminiscent of human CML-CP (Lin−CD34+CD38− LSCs > Lin−CD34+CD38+ LPCs). BCR-ABL1 induction increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA damage (8-oxoG, DSBs) in muLSCs, but not in muLPCs. In addition, CFSEmax/eFluor670max quiescent muLSCs displayed more ROS (superoxide, hydrogen peroxide) and oxidative DNA damage (8oxoG, DSBs) than non-induced counterparts. Currently, we are examining genomic instability in the most primitive long-term muLSCs (Lin−c-Kit+Sca1+CD34−Flt3−). Lastly, single nucleotide polymorphism (SNP) arrays detected a variety of genetic aberrations (addition, deletions) in BCR-ABL1–induced Lin− BM cells. Individual mice displayed a great degree of diversity in the intensity of genetic instability accumulating between 31 to 826 aberrations, which recapitulate heterogeneity of sporadic aberrations detected in CML-CP patients. These aberrations include deletions in Trp53 and Ikzf1, and additions in Zfp423 and Idh1 genes, which have been linked to progression from CML-CP to CML-BP. In summary, by using the SCLtTA/BCR-ABL1 inducible transgenic mouse model of CML-CP we showed that muLSCs, but not muLPCs, displayed elevated levels of ROS-induced oxidative DNA damage likely resulting in the accumulation of extensive genetic aberrations. This observation supports the hypothesis that genomic instability in CML-CP originates in LSCs. Current analysis of microarrays may shed some light on the mechanisms leading to enhanced ROS production and accumulation of oxidative DNA damage in muLSCs. Disclosures: Koschmieder: Novartis, Bristol-Myers Squibb: Consultancy.

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.

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 (CFSEmax) and proliferative (CFSElow) 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 (CFSEmax) 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, CFSEmax and CFSElow 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 CFSElow cells, but also in LSCs-enriched CD34+CD38- and CFSEmax 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. Disclosures: No relevant conflicts of interest to declare.