Targeting the Rac-mitochondrial ROS pathway reduces genomic instability in BCR-ABL1 leukemia cells. (A) GFP⁺ BCR-ABL1-32Dcl3 cells expressing Rac(T17N) and GFP- BCR-ABL1-32Dcl3 control cells (C) as illustrated. BCR-ABL1^+/+ and Rac2^−/− muBMCs are described in Figure 5F (B); BCR-ABL1-32Dcl3 Rho0 cells (Rho0) and BCR-ABL1-32Dcl3 MRC-proficient cells (C) as confirmed by RT-PCR–detected down-regulation of expression of Cox II in Rho0 cells; and BCR-ABL1-32Dcl3 cells expressing MitCat (D), or empty plasmid (E) as documented by Western analysis showing the expression of BCR-ABL1, MitCat, and endogenous catalase (Cat), were maintained for 8 (B) or 12 (A,C,D) weeks in liquid culture. ROS were measured using CC1 and DCFDA fluorescence. The frequency of TKI-resistant (TKIR) clones and/or spectral karyotyping analysis of chromosomal aberrations were determined; results represent mean values of a minimum of 3 experiments ± SD. (E) In the in vitro experiment, BCR-ABL1 muBMCs and CML-CP CD34⁺ cells were cultured either without (−) or with SS20 or SS31 peptides. ROS were measured using CC1 and DCFDA fluorescence; results represent mean values of a minimum of 3 experiments ± SD. In the in vivo experiment, 5 SCID mice/group bearing GFP⁺ BCR-ABL1 muBMCs were treated with SS20 or SS31 for 6-8 weeks. ROS (CC1 fluorescence) and the number of TKIR clones in GFP⁺ muBMCs were determined. (F) Left panel shows 5 SCID mice/group bearing GFP⁺ BCR-ABL1 muBMCs treated with imatinib and SS20 or SS31 for 8 weeks. TKIR clones were detected in GFP⁺ cells from BM and spleen. Right panel shows CD34⁺ cells from 2 CML-CP patients cultured for 6 weeks with imatinib and SS20 or SS31 in medium supplemented with growth factors, after which time the frequency of TKIR clones was determined. *P < .05 compared with control, untreated, and SS20-treated cells or animals.