Figure 4.

Knockdown of Cyb561 diminishes self-renewal of KMT2A::MLLT3-driven leukemia stem cells by enhancing intracellular levels of reactive oxygen species. (A) Frequency of leukemia stem cell-enriched granulocyte-monocyte progenitors (L-GMP) in the bone marrow (BM) from primary recipients injected with 1x10⁵ acute myeloid leukemia (AML) cells transduced with control short hairpin (sh) RNA (shRNA CTR) or Cyb561 shRNA #2, at week 4 after transplantation (N=5). (B) Cell cycle phase distribution of L-GMP cells in BM from primary recipients injected with shRNA CTR or Cyb561 shRNA #2 AML cells, at week 5 after transplantation (N=4). (C) Percentage of apoptotic L-GMP cells in the BM from primary recipients transplanted with shRNA CTR or Cyb561 shRNA #2 AML cells (N=4). (D) Frequency of L-GMP in the BM from recipients receiving AML cells transduced with shRNA CTR or Cyb561 shRNA #2 following treatment with saline or N-acetyl-L-cysteine (NAC) (N=5). (E) Survival analysis of recipient mice receiving AML carrying shRNA CTR or Cyb561 shRNA #2 after treatment with saline or NAC. The median survival of the four groups of recipients was 32, 30, 55, and 35 days after transplantation (P<0.01, Mantel-Cox test, N=6). (F) Diagrammatic model of the miR-30e-5p/Cyb561/ROS signaling pathway. Data are representative of two or three independent experiments. All data are represented as mean ± standard deviation. Two-tailed Student t tests were used to assess statistical significance (N.S.: not significant; *P<0.05; **P<0.01). AsA: ascorbate; GSSG: oxidated glutathione; GSH: reduced glutathione; DHA: dehydroascorbate; ROS: reactive oxygen species.