Increased ROS Stress and Alterations in the Bone Marrow Microenvironment Contribute to DNA Damage in Hematopoietic Stem/Progenitors Carrying a Csf3r Truncation Mutation.

Abstract 3630

Poster Board III-566

Severe congenital neutropenia (SCN) is an inherited disorder of granulopoiesis that is associated with a markedly increased risk of developing MDS/AML. Somatic mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are associated with the development of MDS/AML in SCN. These mutations invariably produce a truncated Csf3r that, though remaining ligand-dependent, transmits a hyperproliferative signal.

Knock-in mice carrying a truncation mutation of the Csf3r (termed d715 G-CSFR) have normal basal granulopoiesis but an exaggerated neutrophil response to G-CSF treatment. We recently reported that expression of the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell level that is dependent upon exogenous G-CSF. Though not sufficient by itself, we previously reported that the d715 G-CSFR was able to cooperate with PML-RARa to induce AML in mice. Herein, we explore mechanisms by which CSF3R truncation mutations contribute to leukemic transformation. Specifically, we test the hypothesis that altered signaling by the d715 G-CSFR contributes to genetic instability through induction of reactive oxygen species (ROS) stress. We show that the basal level of ROS in c-KIT⁺ Sca⁺ lineage⁻ (KSL) hematopoietic stem/progenitor cells (HSPCs) was similar in cells expressing wildtype and d715 G-CSFR. However, 24 hours after in vivo G-CSF stimulation, whereas the level of ROS was not significantly changed in wildtype KSL cells, it was induced 3.4 ± 0.1 fold in d715 G-CSFR cells (p = 0.009). To determine whether this increased ROS stress contributed to DNA damage, levels of phosphorylated histone gH2AX (pH2AX) were measured by flow cytometry. In wildtype mice, short-term (24 -hour) treatment with G-CSF had no affect on pH2AX levels in KSL cells. However, in d715 G-CSFR mice, G-CSF treatment was associated with a modest but significant increase in pH2AX levels (1.9 ± 0.1 fold; p = 0.0007). We and others previously showed that prolonged (more than 3-4 days), but not short-term (24 hour), treatment with G-CSF is associated with disruption of the osteoblast niche in the bone marrow. Since, most patients with SCN are treated chronically with G-CSF, we measured osteoblast activity in the bone marrow and H2AX phosphorylation in KSL cells after 7 days of G-CSF treatment. Notably, osteoblast activity, as measured by CXCL12 and osteocalcin expression, was reduced to a greater extent in d715 G-CSFR versus wild type mice. Interestingly, compared with short-term G-CSF treatment, pH2AX levels were significantly higher in d715 G-CSFR KSL cells after 7 day G-CSF treatment (2.6 ± 0.3 fold ± SEM; p = 0.006). To establish whether induction of ROS was responsible for the increase in pH2AX, we next co-administered the antioxidant N-acetyl cysteine (NAC) during the 7-day G-CSF treatment. As expected, induction of ROS was markedly suppressed in KSL cells by NAC administration. Moreover, the increase in pH2AX levels in d715 G-CSFR KSL cells by G-CSF was completely blocked by NAC administration.

Collectively, these data suggest that both increased ROS stress and altered bone marrow microenvironment may contribute to genomic instability and leukemic transformation in patients with SCN carrying a CSF3R truncation mutation. Moreover, these data raise the possibility that anti-oxidant therapy may be an effective strategy to prevent MDS/AML in SCN.