Investigation of Impact Induced By AML1-ETO on Self-Renewal and Differentiation of Hematopoietic Stem and Progenitor Cells in Pre-Leukemic Stage

AML1-ETO fusion gene is the most common cytogenetic subtype of AML, which occurs in approximately 15% of acute myeloid leukemia (AML) patients. More than 85% of patients could achieve complete remission after induction chemotherapy. However, it was less effective on the patients over the age of 60 who cannot tolerate standard chemotherapy. And nearly 30% of patients relapsed eventually with a dismal outcome. Therefore, identification of the initiating abnormal cells to inhibit the early occurrence induced by AML1-ETO is an urgent problem. In this study, an inducible conditional AML1-ETO knock-in murine model (Aml1Eto/+; Mx1-Cre) was successfully established, hereafter called AEKI. The stage of malignant transformation in hematopoietic stem and progenitor cells (HSPC) was identified. We found that the absolute number of LT-HSCs (1.43×106 vs 4.85×103, p<0.0001), ST-HSCs (7.90×106 vs 1.88×104, p<0.0001) and MPPs (6.07×105 vs 1.82×104, p=0.0358) in bone marrow (BM) increased significantly in AEKI group compared with that of control group, while CMPs (8.13×103 vs 1.83×105, p=0.0029), GMPs (8.48×103 vs 3.12×105, p=0.0026) and MEPs (1.25×104 vs 2.03×105, p=0.001) decreased. It suggested that the activation of AML1-ETO in vivo resulted in excessive accumulation of HSCs and differentiation blockage from HSCs to HPCs. However, AML1-ETO alone was insufficient to induce the onset of overt leukemia after 300-day observation of survival. Furthermore, the characteristics of AEKI HSPCs in the pre-leukemic stage were investigated. Colony forming assay (CFC) was performed to evaluate the stemness of HSCs in vitro. The equivalent of 2×103 LT-HSCs were isolated from BM of each group and plated for 14 days. AEKI LT-HSCs gave rise to dysplastic colonies and lost replating capability compared to that of control group (colony counts: 19.33 vs 30.17, p=0.016). Moreover, a progressive decrease in PB chimerism was observed in AEKI recipients via competitive transplantation assay in vivo (15.8% vs 62.5%, p<0.0001), for which BM cellularity exhibited a significant decrease (7.24×106 vs 4.81×107, p<0.0001). But it led to an notably expansion in HSC number (LT-HSC:1.18×105 v 8.59×103, p=0.0035) which had a same tendency as the phenotype of primary mice. Based on Ki67 and DAPI staining, proportion of HSCs in G1 phase increased (59.85% vs 38.63%, p=0.0007), while that in G0 (33.58% vs 44.2%, p=0.045) and S/G2/M (4.86% vs 13.41%, p=0.0002) phases decreased. These results implied that AML1-ETO induction could reduce the quiescence and self-renewal ability of HSCs obviously in comparison with normal HSCs. Bulk RNA sequencing was performed in LT-HSCs and GMPs sorted from both groups. Genesets related to cell proliferation, cell cycle regulation and Myc targets were enriched in LT-HSCs from control group. It was also found that genesets of tricarboxylic acid (TCA) cycle, glycolysis and fatty acid metabolism were enriched in LT-HSCs from control group. To validate it, Seahorse extracellular flux assay was performed to evaluate oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on HSPC population (c-kitenriched cells). We found that OCR and ECAR both decreased significantly in AEKI group that supported the results from RNA-seq. RNA-seq data also showed that fatty acid metabolic pathway was enriched in AEKI group during the process from LT-HSCs to GMPs, which suggested that AEKI HSPCs maintained abnormal function through excessive fatty acid supply while it was not necessary during normal hematopoiesis. To confirm the above speculation, the mice were fed with fat-free or normal food after the AML-ETO induction in vivo to evaluate the impact of fatty acid on hematopoiesis. Interestingly, BM chimerism increased on the fat-free group (31.56% vs 15.68%, p=0.013), with the absolute number of LT-HSCs (5.16×105 vs 1.80×106, p=0.068), ST-HSCs (1.28×106 vs 2.72×106, p=0.026) and MPPs (4.70×104 vs 2.53×105, p=0.013) reduced obviously compared with in a normal diet. It suggested the excessive accumulation of HSCs was partly recovered. In summary, our study suggested that AML1-ETO induction could cause HSCs to lose self-renewal potential and lead to differentiation disruption during the pre-leukemic stage of AML. Limiting exogenous lipid uptake was able to rescue the phenotype of differentiation disrupt of HSPCs partly and it could be a promising therapeutic strategy.