Delayed Expansion of Murine BCR-ABL1⁺ Leukemia By Genetic and Pharmacological Inhibition of NOX2

Reactive oxygen species (ROS), produced from reduction of oxygen by the myeloid NADPH oxidase (NOX2), are pivotal effector molecules in the innate immune defense against microbes, but imbalance between the generation and detoxification of ROS may be injurious to cell components. The malignant cells of patients with BCR-ABL1⁺ chronic myeloid leukemia (CML) show elevated ROS production, which has been implicated in the genetic instability BCR-ABL1⁺ cells. However, further studies are required to define the role of ROS in leukemogenesis. We have utilized genetically modified murine hematopoietic cells to determine the influence of NOX2-derived ROS for the in vivo expansion of transplanted BCR-ABL1⁺ cells. Irradiated C57BL/6J mice received transplants of wild-type (WT) or Nox2 knockout (Nox2 -KO) hematopoietic cells that were transduced to express GFP-labeled human BCR-ABL1 by use of the p210 construct cloned into a MSCV vector. In vitro experiments using the NOX2 inducer D-peptide confirmed that Nox2 -KO BCR-ABL1⁺ cells were devoid of ROS production via NOX2. Peripheral blood was drawn biweekly from mice receiving WT and Nox2 -KO transplants for fluorimetric quantification of BCR-ABL1⁺ cells. We observed prolonged survival for mice receiving Nox2 -KO vs. WT BCR-ABL1 transplants (n=43, p=0.017, logrank test) along with a trend towards reduced expansion of leukemic cells in vivo . To determine effects of pharmacological inhibition of NOX2 on leukemic expansion, the NOX2 inhibitor histamine dihydrochloride (HDC) was administered to mice carrying WT or Nox2 -KO BCR-ABL1⁺ transplants. In vitro experiments confirmed that HDC reduced NOX2-dependent induction of ROS formation in WT BCR-ABL1⁺ cells. Treatment of age-matched mice with HDC (1.5 mg three times per week i.p.) delayed the expansion of WT BCR-ABL1⁺ cells at four weeks after transplantation (n=18, p<0.05, t-test) but did not reduce the burden of leukemia in mice receiving Nox2 -KO BCR-ABL1⁺ transplants. Initial experiments using primary human CML cells revealed differential expression of redox-related genes in CD34⁺CD38^- leukemic stem cells vs. cord blood stem cells. Our results implicate NOX2 as a conceivable therapeutic target in BCR-ABL1⁺ leukemia.