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- Acute myeloid leukemia (AML) (1)
- CRISPR-Cas9 (1)
- FLT3-ITD (1)
- NADPH oxidases (NOX) (1)
- Nox4 (1)
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- Reactive oxygen species (ROS) (1)
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Purpose
Oxidative stress has been linked to initiation and progression of cancer and recent studies have indicated a potential translational role regarding modulation of ROS in various cancers, including acute myeloid leukemia (AML). Detailed understanding of the complex machinery regulating ROS including its producer elements in cancer is required to define potential translational therapeutic use. Based on previous studies in acute myeloid leukemia (AML) models, we considered NADPH oxidase (NOX) family members, specifically NOX4 as a potential target in AML.
Methods
Pharmacologic inhibition and genetic inactivation of NOX4 in murine and human models of AML were used to understand its functional role. For genetic inactivation, CRISPR-Cas9 technology was used in human AML cell lines in vitro and genetically engineered knockout mice for Nox4 were used for deletion of Nox4 in hematopoietic cells via Mx1-Cre recombinase activation.
Results
Pharmacologic NOX inhibitors and CRISPR-Cas9-mediated inactivation of NOX4 and p22-phox (an essential NOX component) decreased proliferative capacity and cell competition in FLT3-ITD-positive human AML cells. In contrast, conditional deletion of Nox4 enhanced the myeloproliferative phenotype of an FLT3-ITD induced knock-in mouse model. Finally, Nox4 inactivation in normal hematopoietic stem and progenitor cells (HSPCs) caused a minor reduction in HSC numbers and reconstitution capacity.
Conclusion
The role of NOX4 in myeloid malignancies appears highly context-dependent and its inactivation results in either enhancing or inhibitory effects. Therefore, targeting NOX4 in FLT3-ITD positive myeloid malignancies requires additional pre-clinical assessment.
Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.