Chronic Myeloid Leukemia Stem Cells (LSCs) and Leukemia Progenitor Cells (LPCs) Display Overlapping and Unique Mechanisms of Genomic Instability: The Role of PI3k-AKT and PI3k-Rac2-PAK Pathways

BCR-ABL1 fusion tyrosine kinase transforms hematopoietic stem cells (HSCs) and cause chronic myeloid leukemia in chronic phase (CML-CP), which is a stem cell (leukemia stem cell=LSC) -derived but a progenitor (leukemia progenitor cell=LPC)-driven disease. Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, nilotinib and ponatinib revolutionized the treatment of CML.

We showed that reactive oxygen species (ROS) may increase the levels oxidative DNA damage in TKI-naïve and TKI-treated Lin-CD34+CD38- LSCs, including quiescent LSCs, and in Lin-CD34+CD38+ LPCs. Since DNA repair mechanisms display reduced fidelity/activity in CML cells, overproduction of ROS may promote genomic instability (accumulation of point mutations and chromosomal aberrations) resulting in resistance to TKIs and malignant progression of the disease to accelerated (CML-AP) or blast (CML-BP) phase.

BCR-ABL1 kinase stimulates numerous signaling pathways to induce and maintain transformation of hematopoietic cells. We and others found that phosphatidylinositol-3 kinase (PI3k) plays an essential role not only in growth factor independent proliferation and protection from apoptosis of CML cells, but also in overproduction of mitochondrial ROS. Here we investigated the role of PI3k downstream effectors, AKT serine/threonine kinase and Rac GTPase in genomic instability in TKI-naïve and TKI-treated LSCs and LPCs.

Using CML-CP primary cells and an inducible model of murine CML-CP –like disease we determined that Rac2 – PAK serine/threonine pathway alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III thereby generating high levels of ROS in TKI-naïve and TKI-treated LSCs and LPCs. In addition, AKT appeared to play an essential role in generation of ROS-induced oxidative DNA damage in TKI-naïve and TKI-treated LPCs, but not in LSCs. Inhibition of AKT did not affect the activity of Rac2 and inhibition of Rac2 did not affect AKT, clearly indicating that their activation status does not depend on each other. Targeting Rac2 and AKT either by mutations/deletions/shRNAs or small molecule inhibitors/peptides reduced genomic instability resulting in diminished frequency of TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations.

Altogether TKI-naive and TKI-treated LSCs (including quiescent LSCs) display only PI3k-Rac2-PAK pathway, whereas LPCs contain PI3k-Rac2-PAK and PI3k-AKT pathways responsibble for ROS-induced oxidative DNA damage and genomic instability. Our findings may have more broad application because other oncogenic tyrosine kinases expressed in hetatopoietic malignancies induce similar mechanisms of genomic instability.