Nrf2 Deficiency Leads to Altered Hematopoietic Stem Cell Function and Increased Sensitivity to Alkylating Agent Induced Myeloid Dysplasia,

Abstract 3828

Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a lethal complication of cancer treatment. In a previous study we conducted gene expression analysis comparing peripheral blood stem cell (PBSC) CD34+ cells from patients who subsequently developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) and matched controls that did not develop t-MDS/AML after aHCT. This study showed that the Nrf2-mediated oxidative stress response was one of the top ranked canonical pathways downregulated in t-MDS/AML cases compared to controls. Nrf2 is a basic region-leucine-zipper transcription factor that regulates expression of numerous detoxifying and antioxidant genes as well as ubiquitination and proteasomal degradation proteins. Since the Nrf2 pathway is a major cellular defense mechanism against oxidative and xenobiotic stresses, we hypothesized that downregulated Nrf2 response may cause increased toxicity after genotoxic therapeutic exposures and contribute to development of t-MDS/AML. To test this hypothesis, we studied Nrf2 knockout mice to determine the effects of Nrf2 deficiency on hematopoiesis under physiological conditions and after exposure to genotoxic stress. We did not observe significant differences in peripheral blood (PB) counts between Nrf2 knockout (KO) mice and wild-type (WT) mice. Analyses of hematopoietic cells from PB, bone marrow (BM) or spleen using multi-color flow cytometry demonstrated that the compositions of granulocyte, B cell and T cell lineages were similar between KO and WT mice. No differences in BM long-term hematopoietic stem cell (LT-HSC, Lin-c-Kit+Sca-1+ Flt3- CD48-CD150+), multi-potent progenitor (MPP, Lin-c-Kit+Sca-1+ Flt3- CD48+), common-myeloid progenitor (CMP, Lin-c-Kit+Sca-1-CD34+CD16/32-), granulocyte/monocyte progenitor (GMP, Lin-c-Kit+Sca-1-CD34+CD16/32+), or megakaryocyte/erythroid progenitor (MEP, Lin-c-Kit+Sca-1-CD34-CD16/32-) populations were seen between KO and WT mice. However, analysis of LT-HSC function using competitive repopulation assays demonstrated significantly reduced chimerism of donor derived cells in PB of recipient mice at 12 weeks and 16 weeks in KO group compared to WT control (42.49±3.09% vs. 54.75±2.13% at 12 weeks and 42.5±2.8% vs. 57.2±3.3% at 16 weeks for KO and WT respectively). Although we did not see significant differences in chimerism of donor-derived hematopoietic stem/progenitor cells in the BM of KO and WT groups at 16 weeks, we did observe significantly reduced contribution of donor-derived cells from the KO group compared to WT control in PB in secondary recipients as early as 8 weeks after secondary transplantation of BM cells (16.5±1.7% vs 61.9±1.5% for KO and WT respectively). These data indicate that Nrf2 deficiency causes cell-intrinsic functional defects in HSC self-renewal. In order to test if Nrf2 deficient hematopoietic cells are more sensitive to genotoxic stress compared to WT cells, we treated KO and WT mice with the alkylating agent ENU and monitored for hematological changes in PB. We did not observe significant differences in hematopoietic recovery after exposure to acute genotoxic stress between KO and WT mice. However, KO mice developed severe anemia at a significantly increased rate and with shorter latency compared to WT mice after ENU treatment (88% in KO vs. 28% in WT at 4 months, p=0.003). Anemic mice developed massive splenomegaly, and histological analysis showed loss of follicular structure and dramatic increase in erythropoiesis with areas of myeloid elements. Pathological examination of BM from anemic mice revealed normal cellularity but reduced erythropoiesis with expansion of myeloid cells. FACS analyses revealed expansion of myeloid (CD11b+, Gr-1+) and immature erythroid population (CD71+Ter119+/dim) in the spleen and BM, and increased CD11b+, Gr-1+ cells in PB. These findings are consistent with development of myeloid dysplasia in these mice. In conclusion, Nrf2 deficiency results in alterations in HSC self-renewal capacity under physiological condition, and leads to increased sensitivity to alkylator-induced myeloid dysplasia in mice.