Both in vitro and in vivo, a DNMT1 depleting, but non-cytotoxic, dose and schedule of decitabine was nonetheless able to induce cell cycle exit in AML cells. The absence of early apoptosis or phosphorylation of p53, efficacy in p53- and p16/CDKN2A-null backgrounds, the major increase in CEBPε and p27/CDKN1B, increase in the myeloid membrane differentiation markers CD11b or CD14, and cellular differentiation, were consistent with p53-independent differentiation mediated cell cycle exit. Hence, this approach to treatment could provide a useful alternative or complement to conventional apoptosis-based therapy. The efficacy of this treatment in vivo against cytarabine resistant p53-null cells and primary cells from a patient with refractory/relapsed AML, supports the translational possibilities.
Why does DNMT1 depletion, or histone deacetylase inhibition, induce differentiation of AML cells? One insight comes from experiments with normal HSC. In normal HSC, DNMT1 depletion by shRNA or by decitabine maintains stem cell phenotype even in differentiation promoting conditions, by preventing repression of key stem cell genes by the differentiation stimuli (26
). However, after the repression of stem cell genes that occurs with lineage-commitment, decitabine can augment expression of late differentiation genes and accelerate differentiation instead (52
). Therefore, baseline differentiation stage is a major determinant of the cell fate response to decitabine treatment. The earliest studies suggested that AML cells with leukemia initiating capacity (measured by xenotransplantation) had a surface phenotype resembling that of normal HSC (CD34+38−) (48
). This suggested that AML cell populations might recapitulate the hierarchical structure of normal hematopoiesis, with a small sub-set of AML cells with a stem cell phenotype sustaining the bulk cell population (53
). Recently, it has been reported that the antibodies used to sort for CD38+ cells can inhibit proliferation and might have technically influenced the earliest studies (54
). Accordingly, in a number of other studies, AML initiating cells had a surface phenotype suggesting lineage-commitment (CD34+38+, CLL-1+, CD71+, CD90 -, c-Kit -) (54
). Cross-species barriers also impact leukemia or cancer initiating capacity (61
). With use of more immuno-compromised mice, or mice which express human cytokines, AML initiating cells have surface features of lineage-commitment (61
). Differentiation absolutely requires and is driven by lineage-specifying transcription factors such as CEBPA. AML cells, including CD34+ and CD34+38− subsets express high levels of CEBPA, but relatively low levels of the key late differentiation driver CEBPE (submitted manuscript). Similarly, we have noted that the promoter CpG methylation profile of MDS and AML cells is consistent with partial differentiation. It could be the partial differentiation of AML cells at baseline, suggested by surface phenotype, lineage-specifying transcription factor expression and promoter CpG methylation patterns, contributes to the contrasting differentiation responses of normal HSC and AML cells to non-cytotoxic DNMT1 depletion.
Although this treatment can bypass the p53-dependence of conventional cytotoxic treatment, it is still limited by the pharmacologic properties of decitabine. The S-phase specific mechanism of action of decitabine was underlined by the correlation of AML cell line sensitivity with KI67 expression (a measure of growth fraction). Therefore, scheduling and duration of exposure is a critical determinant of treatment efficacy. However, decitabine is rapidly metabolized by ubiquitously expressed cytidine deaminase (rapid metabolism by cytidine deaminase, which is highly expressed in the liver, could explain why the liver was a sanctuary site for liver tropic THP1 cells from the effects of cytarabine and decitabine). Hence, the in vivo
half-life of decitabine after IV push is in the order of minutes, compared to many hours in vitro
). An obvious mechanism for treatment failure therefore is that some or many AML cells may complete S-phase while decitabine is absent in vivo
. Since decitabine was originally developed to induce apoptosis in malignant cells (68
), doses to treat AML were escalated to maximum tolerated levels (up to 80 mg/kg infused over 36–44 hours), requiring many subsequent weeks without treatment to allow for recovery from cytotoxic side-effects (64
). A role for differentiation in decitabine-induced cell cycle exit of leukemia cells does not exclude a role for activation of apoptosis genes, anti-metabolite effects, and apoptotic cell cycle exit: these could be the dominant consequences of treatment in some malignant cells, and certainly with higher dose (19
). However, a decrease in the dose (to 15 mg/m2
infused over 3 hours 3X/day on day 1–3, repeated every 6 weeks) led to United States Food and Drug Administration (FDA) approval of decitabine as a treatment for MDS (69
). A further decrease in the daily dose and administration more frequently (20 mg/m2
infused over 1 hour 1X/day on Day 1–5, repeated every 28 days, has further improved MDS treatment clinical results (69
). The results here provide a biological rationale to continue this towards smaller dose and more frequent administration: very low drug levels are sufficient to deplete DNMT1, and the decrease in toxicity can be used to administer treatment 1-3X/week for months or years, to capture AML cells entering S-phase asynchronously, and decrease treatment-free intervals that allow unimpeded AML cell division (figure S2
). Clinical trials in the hemoglobinopathies have shown that decitabine 0.2 mg/kg (~7.5 mg/m2
) administered SC has non-cytotoxic DNA hypomethylating and differentiation modifying effects, and can be administered safely from 1–3 X/week (25
These in vitro and in vivo results provide a rationale for adjusting in vivo dose, schedule and route of administration of decitabine to emphasize a non-cytotoxic, normal HSC sparing, p53-independent mechanism of action. Although pharmacologic barriers to optimal clinical translation remain, these can potentially be addressed through further pre-clinical and clinical investigation.