For these studies, we used a murine transgenic mouse model in which PML-RARA is expressed in myeloid cells under the MRP8 promoter. PML-RARA transgenic mice develop impaired myeloid differentiation early in life and APL, with a penetrance of 64% and a median latency of 8.5 months. APL that develops in these transgenic mice fully recapitulates the features of the human disease, including achieving remissions when treated with retinoic acid, which is the drug of choice in this disease. Moreover, these features are retained when preleukemic bone marrows are transplanted in recipient mice.6,15
Thus, this mouse model of APL has attractive features to test preclinical chemopreventive and therapeutic strategies.
We generated cohorts of mice transplanted with bone marrow cells obtained from preleukemic APL transgenic mice to perform our chemoprevention studies. Results are summarized in . Body weights and blood counts were comparable and within the normal range for all treatment groups until leukemia occurred (data not shown). Nonleukemic deaths, unless stated otherwise, were due to acute events secondary to manipulations that are required for venipuncture. We noted no significant drug-related toxic events.
Figure 1. Chronic treatment with 5-azacytidine (5-AC) increases acute promyelocytic leukemia (APL) leukemogenesis. (A) Summary of the mice census. Columns indicate the treatments administered, the cause of death, and the number of mice sacrificed at the study end (more ...)
Significantly, we observed 9 APL cases in the 5-AC treatment group. The mean onset of 5.9 months posttransplantation was strikingly accelerated as compared to the 10 months of the PBS injection group (). There were 7 nonleukemic deaths in this group, 2 of which were due to sarcoma. We have also observed a substantial but nonsignificant APL acceleration in the WD group when compared to the control diet group (P = 0.08) ().
Surprisingly, we observed no difference in leukemia-free survival between the ATRA and the control diet groups (). In addition, 2 of the leukemias that arose in this group were proven to be insensitive to ATRA treatment (data not shown). We found no significant morphological and phenotypic differences between the APL that arose in the various treatment groups (-; note increased percentage of doubly positive Gr1 and Mac1 cells in F-H as compared to E, and data not shown).
To identify at the molecular level the determinants of the APL acceleration in 5-AC–treated mice, we examined the RNA expression profiles of the APL cases that arose in 5-AC–treated mice and in their control group. Among the top 100 most differentially expressed genes, we found several cancer genes down-regulated in the leukemias that arose during treatment with 5-AC such as DMTF1 (a cyclin D–binding protein that exerts a growth-suppressive function), FOXO1A (a tumor suppressor gene), Bach2 (a growth suppressor), DUSP1 (a down-regulator of multiple MAPK signaling pathways and protein kinases that play a role in cell cycle regulation), and sestrin 1 (a transcriptional target of the tumor suppressor p53). Among the genes up-regulated in the leukemias that arose during 5-AC treatment were Birc1e (a IAP inhibitor that antagonizes apoptosis), Sept9 (a proposed growth suppressor), and angiopoietin-like 4 (a gene with prometastatic functions) (Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/entrez
) ( and Suppl. Table S1). When analyzed with the DAVID Bioinformatics Resource and Ingenuity Systems Pathway Analysis tools, these genes cluster in functional classes involved in the regulation of transcription, adaptive immune responses, and apoptosis. Therefore, it is tempting to speculate that these processes are involved with the mechanism(s) causing APL acceleration in 5-AC–treated mice. However, the relevance of these gene alterations versus the effects of 5-AC on APL is yet to be established.
Differential Cancer Gene Expression in APL Leukemias that Arose in 5-AC Treated Mice
Our findings allow us to draw the following conclusions: 1) Chronic 5-AC treatment resulted in a striking acceleration of APL leukemogenesis. This result is surprising given the fact that the PML-RARA oncoprotein induces hypermethylation and transcriptional silencing at its target promoter sites and that this activity contributes to its leukemogenic potential.11
DNA hypomethylation has been associated with chromosomal instability, reactivation of transposable elements, loss of imprinting, and activation of proto-oncogenes.25
We speculate that 5-AC may cause genome-wide genetic and epigenetic effects that promote leukemogenesis. For example, recent reports show that genomic hypomethylation causes genomic instability and lymphomagenesis in mice.23,24
2) Chemoprevention with ATRA did not prevent APL leukemogenesis and instead results in ATRA-resistant APL, a known complication of ATRA therapy.21,22
3) WD induced a trend toward APL acceleration that, although did not reach statistical significance (P
= 0.08), suggests that WD may represent a risk factor for APL leukemogenesis. Further, because the effects of WD appear to parallel those by 5-AC, we may infer that these effects are due, in large measure, to its enhanced hypomethylating potential.
Our findings have in turn important clinical implications and suggest that further investigations should be considered to address the long-term effects of ATRA and 5-AC in APL and myelodysplastic syndromes. The study of the function of the genes differentially expressed between the 5-AC–treated and control mice may provide insights into the mechanisms underlying 5-AC–induced leukemogenesis.