Cell Cycle Entry Potentiates Elimination of Chemotherapy-Resistant Human AML Stem Cells.

Abstract 1422

Poster Board I-445

Acute myeloid leukemia (AML) is associated with poor long-term prognosis despite advances in therapeutic modalities over the past few decades. As leukemia stem cells (LSCs) capable of AML initiation may contribute to recurrent disease, LSC-targeted therapies are required to overcome disease relapse and to improve long-term patient outcomes. We previously reported that human AML CD34+CD38- cells self-renew, generate non-stem leukemic cells, and possess potential to initiate leukemia following engraftment of newborn NOD/SCID/IL2rgKO mice. In the recipient bone marrow (BM), AML LSCs were found to reside preferentially within the endosteal region and exhibited chemotherapy resistance. In addition, we observed that AML cells abutting the BM endosteum were cell cycle quiescent while AML cells in the center of the BM were cycling. Based on these findings, we hypothesized that induction of cell cycle entry in quiescent AML LSCs may increase their susceptibility to chemotherapeutic agents, leading to enhanced elimination of LSCs. To test this hypothesis, we assessed the effect of granulocyte colony-stimulating factor (G-CSF) on cell cycle status and chemotherapy susceptibility of primary human AML LSCs in vivo using the NOD/SCID/IL2rgKO xenotransplantation model. In AML-engrafted recipient mice transplanted with LSCs from seven AML patients, flow cytometric analyses demonstrated a significant reduction of quiescent LSCs following 300μg/kg G-CSF sc daily for 5 days (%G0 within hCD34+CD38- BM cells (mean+/-s.e.m): 49.2+/-2.6 (n=47) and 20.5+/-2.0 (n=36), control and G-CSF treated recipients, respectively, p<0.0001 by two-tailed t test). Direct examination of recipient BM in situ revealed cell cycle entry of human AML cells abutting the BM endosteum as evidenced by increased Ki67 expression. Next we developed an in vivo treatment model evaluating the effect of cell cycle induction on chemotherapy-responsiveness of human primary AML LSCs. Human AML-engrafted recipients received AraC alone (1g/kg ip daily for 2 days) or G-CSF followed by AraC (300μg/kg G-CSF sc daily for 5 days with 1g/kg AraC ip daily on days 4 and 5). The proportion of viable active caspase 3-negative human LSCs decreased significantly with pre-chemotherapy cell cycle induction (% active caspase 3-negative hCD34+CD38- BM cell (mean+/-s.e.m.): 82.7+/-1.3% (n=33) and 40.4+/- 3.1% (n=30), AraC alone- and G-CSF followed by AraC-treated recipients, respectively, p<0.0001 by two-tailed t test). TUNEL staining of the recipient BM showed increased apoptosis of AML cells abutting the BM endosteum in recipients receiving AraC following cell cycle induction. Limiting dilution serial transplantation of residual viable human AML cells in the BM of treated recipients showed 100-fold reduction in the frequency of LSCs capable of initiating AML in secondary recipients (BM LSC frequency: 1/560 (n=125) and 1/55,076 (n=109), AraC alone- and G-CSF then AraC-treated recipients, respectively, p=0.0001 by two-tailed t test). At 24 weeks post-transplantation, 89.4% of secondary recipients of G-CSF followed by AraC-treated mice survived compared with only 2.0% survival in secondary recipients of AraC alone-treated mice (p<0.0001, survival estimated by Kaplan-Meier method). These findings indicate that cell cycle status is a key determinant of LSC chemo-responsiveness and that therapeutic strategies promoting LSC cell cycle entry may improve outcomes in AML.