p53 independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid leukemia.
ABSTRACT Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo regimens of the deoxycytidine analogue decitabine that deplete the chromatin-modifying enzyme DNA methyl-transferase 1 without phosphorylating p53 or inducing early apoptosis were determined. These decitabine regimens but not equimolar DNA-damaging cytarabine upregulated the key late differentiation factors CCAAT enhancer-binding protein ɛ and p27/cyclin dependent kinase inhibitor 1B (CDKN1B), induced cellular differentiation and terminated AML cell cycle, even in cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xenotransplanted AML cells was abrogated but normal hematopoietic stem cell engraftment was preserved. In vivo, the low toxicity allowed frequent drug administration to increase exposure, an important consideration for S phase specific decitabine therapy. In xenotransplant models of p53-null and relapsed/refractory AML, the non-cytotoxic regimen significantly extended survival compared with conventional cytotoxic cytarabine. Modifying in vivo dose and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to standard therapy.
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ABSTRACT: Although uncontrolled proliferation is a distinguishing property of a tumor as a whole, the individual cells that make up the tumor exhibit considerable variation in many properties, including morphology, proliferation kinetics, and the ability to initiate tumor growth in transplant assays. Understanding the molecular and cellular basis of this heterogeneity has important implications in the design of therapeutic strategies. The mechanistic basis of tumor heterogeneity has been uncertain; however, there is now strong evidence that cancer is a cellular hierarchy with cancer stem cells at the apex. This review provides a historical overview of the influence of hematology on the development of stem cell concepts and their linkage to cancer.Blood 01/2009; 112(13):4793-807. · 9.06 Impact Factor
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ABSTRACT: Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.Nature 03/1994; 367(6464):645-8. · 38.60 Impact Factor
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ABSTRACT: The nucleoside analog 5-azacytidine (5-aza-CR) induced marked changes in the differentiated state of cultured mouse embryo cells and also inhibited the methylation of newly synthesized DNA. The DNA strand containing 5-aza-CR remained undermethylated in the round of DNA synthesis following analog incorporation. The extent of inhibition of DNA modification and induction of muscle cells in treated cultures were dependent on the 5-aza-CR concentration over a narrow dose range. Experiments with the restriction enzyme Hpa II, which is sensitive to cytosine methylation in the sequence CCGG, demonstrated that the DNA synthesized in 5-aza-CR-treated cultures was maximally undermethylated 48 hr after treatment. Three other analogs of cytidine, containing a modification in the 5 position of the pyrimidine ring [5-aza-2'-deoxycytidine(5-aza-CdR), pseudoisocytidine (psi ICR) and 5-fluoro-2'-deoxycytidine(FCdR)] also induced the formation of muscle cells and inhibited DNA methylation. In contrast, 1-beta-D-arabinofuranosylcytosine (araC) and 6-azacytidine (6-aza-CR) did not inhibit DNA methylation or induce muscle formation, whereas 5-6-dihydro-5-azacytidine (dH-aza-CR) was a poor inducer of muscle cells and a poor inhibitor of DNA methylation. These results provide experimental evidence for a role for DNA modification in differentiation, and suggest that cytidine analogs containing an altered 5 position perturb previously established methylation patterns to yield new cellular phenotypes.Cell 06/1980; 20(1):85-93. · 31.96 Impact Factor
TITLE: p53 independent epigenetic-differentiation treatment in xenotransplant models of acute myeloid
Running title: p53 independent treatment for AML
Authors and Institutions: Kwok Peng Ng1 *, Quteba Ebrahem1 *, Soledad Negrotto1, Reda Z. Mahfouz1,
Kevin A. Link3, Zhenbo Hu1, Xiaorong Gu1, Anjali Advani 2, Matt Kalaycio 2, Ronald Sobecks 2, Mikkael
Sekeres 2, Edward Copelan 2, Tomas Radivoyevitch 3, Jaroslaw Maciejewski1, 2, James C. Mulloy4, Yogen
* These authors contributed equally to the work
1 Department of Translational Hematology & Oncology Research, Taussig Cancer Institute, Cleveland Clinic,
2 Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic,
3 Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, USA
4 Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital, Cincinnati, USA
Correspondence: Yogen Saunthararajah, MD, Taussig Cancer Institute, 9500 Euclid Avenue R40, Cleveland,
OH 44195, tel: 216 444 8170, email: firstname.lastname@example.org
Word and other counts:
Abstract: 181 words
Text (introduction, materials and methods, results and discussion): 4,077 words
Tables/Figures: 6 figures
Supplementary material: 5 supplementary figures
References: 61 references
Suppression of apoptosis by TP53 mutation contributes to resistance of acute myeloid leukemia (AML) to
conventional cytotoxic treatment. Using differentiation to induce irreversible cell cycle exit in AML cells could be
a p53-independent treatment alternative, however, this possibility requires evaluation. In vitro and in vivo
regimens of the cytosine analogue decitabine that deplete the chromatin modifying enzyme DNA methyl-
transferase 1 (DNMT1) without phosphorylating p53 or inducing early apoptosis were determined. These
decitabine regimens but not equimolar DNA-damaging cytarabine up regulated the key late differentiation
factors CEBPε and p27/CDKN1B, induced cellular differentiation, and terminated AML cell-cycle, even in
cytarabine-resistant p53- and p16/CDKN2A-null AML cells. Leukemia initiation by xeno-transplanted AML cells
was abrogated but normal hematopoietic stem cell (HSC) engraftment was preserved. In vivo, the low toxicity
allowed frequent drug administration to increase exposure, an important consideration for S-phase specific
decitabine therapy. In xeno-transplant models of p53-null and relapsed/refractory AML, the non-cytotoxic
regimen significantly extended survival compared to conventional cytotoxic cytarabine. Modifying in vivo dose
and schedule to emphasize this pathway of decitabine action can bypass a mechanism of resistance to
Although conventional chemotherapeutics for acute myeloid leukemia (AML) can have differing proximal
mechanisms of action, such as topoisomerase inhibition or termination of DNA chain synthesis, a final common
pathway converges onto p53, a stress and DNA damage sensor, and a master regulator of apoptosis
(reviewed in (1)). Therefore, mutation and chromosome deletion at the TP53 locus is associated with treatment
resistance both in vitro (2;3) and in vivo: partial or complete remissions in response to intensive chemotherapy
were achieved in 81% of AML cases without, and 33% of cases with TP53 mutations (4). Similarly, responses
in patients with myelodysplastic syndrome (MDS) treated with intensive chemotherapy or low dose cytarabine
were 60% of cases without, and 8% of cases with TP53 mutations (4). Even in cases in which TP53 itself is not
directly mutated or deleted, the p53 pathway may be targeted by genetic abnormalities in p53 cofactors (5): in
blast transformed myeloproliferative disease, which responds only transiently if at all to conventional therapy,
45.5% of cases had either TP53 defects or gain of MDM4 (6). In MDS and AML cases with complex
cytogenetic abnormalities, another group of patients with very poor treatment outcomes, the rate of TP53
mutation can exceed 70% (7).
Hence, especially for certain sub-types of MDS and AML, there is a need for treatment that is not mediated
through p53 and apoptosis. Interestingly, although p53-null mice are cancer prone, the development of these
mice is essentially normal, with normal patterns of differentiation in almost all tissues (reviewed in (8)). This
suggests that differentiation-mediated cell cycle exit is usually p53-independent. Using differentiation to
terminate cancer cell proliferation was proposed more than 50 years ago (9-11), and differentiation has been
observed in AML and cancer cells treated with drugs that inhibit chromatin modifying enzymes, such as histone
deacetylase inhibitors (HDACi), and 5-azacytidine and decitabine that deplete DNA methyl-transferase 1
(DNMT1) (12-18). However, the effects of HDACi are not confined to histones; HDACi can alter the acetylation
status of structural, signaling and transcription factor proteins, producing wide-spread cellular effects including
apoptosis. 5-azacytidine and decitabine are cytosine analogues, and at high concentrations, can cause DNA
damage that triggers apoptosis. It is also possible that the chromatin-modifying effects of these drugs renew
expression of tumor suppressor genes that mediate apoptosis. Hence, it has not been clear that differentiation-
mediated cell cycle exit is the most important therapeutic action of these compounds (reviewed in (19)). There
has even been debate about whether inhibition of chromatin-modifying enzymes is the most important effect of
these drugs (19). Therefore, if such treatment is to bypass a major limitation of conventional therapy,
demonstration of a p53-independent mechanism of action, then rationalization of dose and schedule to
emphasize this pathway of action, and demonstration of in vivo efficacy, are important translational challenges.
Unlike the cytosine analogues cytarabine or gemcitabine, the sugar back-bone of decitabine is unmodified.
Therefore, at low concentrations, decitabine can incorporate into the newly synthesized DNA strand during S-
phase without terminating chain elongation (20;21). These non-DNA damaging, non-cytotoxic concentrations
of decitabine can nonetheless deplete DNMT1, both in vitro and in vivo (12;20-24). Our first objective was to
determine if these concentrations of decitabine, which do not kill normal hematopoietic stem cells (HSC) (low
concentrations of decitabine have increased normal hematopoietic stem cell self-renewal in a number of
studies (25-28)), and which may not induce early apoptosis, can nonetheless induce irreversible cell cycle exit
in AML cells. Cell cycle exit is mediated by a family of highly conserved cyclin dependent kinase inhibitors
(CDKN): p16/CDKN2A is implicated in apoptotic cell cycle exit, whereas p27/CDKN1B is known to mediate cell
cycle exit with differentiation (29-32). Therefore, our second objective was to measure changes in CDKN
protein expression in response to decitabine and equimolar cytarabine, to differentiate between apoptosis and
differentiation-mediated cell cycle exit, and to determine if these changes were dependent on p53. In vitro
experiments were conducted in p53 wild-type and p53-null MLL-AF9 AML cells. The effects of treatment on
p53 upregulation and phosphorylation could be measured in p53 wild-type cells, whereas p53-null cells could
be used to confirm p53-independence of observed effects. Finally, we examined if a decitabine dose, schedule
and route of administration rationalized for a non-cytotoxic mechanism of action in vivo, could be efficacious in
murine xenotransplantation models of p53-null and relapsed/refractory human AML, to evaluate the
translational potential of this alternative, non-cytotoxic treatment approach.
Healthy volunteer and patient samples: Umbilical cord blood was collected during normal full-term deliveries,
and bone marrow aspirates were collected from AML patients and healthy volunteers. All collections occurred
after written informed consent of the mother, patient or volunteer as per Case Western Reserve University and
Cleveland Clinic IRB approved protocols. Anonymized clinical hematopathology data was associated with
Isolation of CD34+ cells: CD34+ cells from umbilical cord blood or bone marrow aspirates were purified using a
magnetic cell sorting system (CD34 MicroBead Kit #130-046-702, Miltenyl Biotec Inc, Auburn, CA) according
to manufacturer instructions. The purity of the CD34+ population (typically from 95 to 99%) was determined by
flow cytometry with a FITC-conjugated monoclonal antibodies against CD34 (Clone 581, Beckman Coulter,
Miami, FL, USA).
AML cells analyzed: Three types of AML cells were analyzed – (i) p53 wild-type MLL-AF9 cells were generated
as described (33). These cells have a gene-expression profile similar to primary human MLL-AF9 leukemia
cells (33), and self-renew indefinitely both in vitro and in vivo, initiating invasive AML in transplanted mice (33).
(ii) p53-null THP1 cells were purchased from ATCC (Manassas, VA). This morphologically monocytoid AML
cell line (M5 morphology) contains an MLL-AF9 fusion and is homozygously mutated at the TP53 and
CDKN2A loci. THP1 cells used for xeno-transplantation were transfected to express luciferase. (iii) Primary
(fresh) AML cells from a patient with relapsed/refractory AML. These cells had a myelomonocytic morphology
(M4) and by standard metaphase karyotyping contained t(8;18)(q22:q23) and t(11;13)(q21:q12).
Human hematopoietic cell culture: Normal human hematopoietic cells and p53 wild-type MLL-AF9 cells were
cultured in IMDM supplemented with 10% fetal bovine serum and 10ng/ml of the following human cytokines:
stem cell factor (SCF), FLT3 ligand (FLT3), thrombopoietin (TPO), interleukin-3 (IL-3) and interleukin-6 (IL-6).
THP1 cells were cultured in RPMI 1640 media without cytokine supplementation.
Treatment of cells with decitabine: Decitabine stock solution (5 mM) was generated by reconstituting lyophilized
decitabine in 100% methanol. Stock solution was stored at -20 0C for up to 3 weeks. Similar amounts of methanol
are added to untreated control cells. For in vitro experiments, cells were treated with decitabine (0.5 µM) on day 1
and 4 unless otherwise specified. For in vitro treatment followed by transplantation of cells into mice by tail-vein
injection, cells were treated with decitabine 0.5 µM on day 1, 0.2 µM on day 2, 0.5 µM on day 5, 0.2 µM on day 6,
and transplantation on day 7.
Apoptosis detection: Apoptosis was detected by Annexin-V and 7AAD or PI co-staining using the APOAF
commercial kit (Sigma).
Clonogenic progenitor assays: p53 wild-type and p53 null MLL-AF9 cells in liquid culture were treated with
decitabine or Cytarabine 0.5 µM on day 1 and 4. On day 5, identical numbers of cells from treated and untreated
control cultures were plated in decitabine free-semisolid media (MethoCult H4434, Stem Cell Technology, 2000
cells/ml). Colony-forming units (CFU) were identified by morphology and counted under an inverted microscope at
10 days post-plating.
SDS-PAGE and Western Blotting: Approximately 100 µg of cytoplasmic and nuclear protein extracts from cells,
together with molecular weight markers, were subjected to SDS-PAGE on 4-12% gradient gels (Invitrogen)
followed by transfer to PVDF membranes (Invitrogen). Blots were probed using antibodies for DNMT1 (Abcam
ab16632), Phospho-p53 (Cell Signaling #9286), p53 (Sigma P6874), p15 (Cell Signaling #4822), p21 (Cell
Signaling #2946), p16 (Santa Cruz Biotech sc-81613 ; Cell Signaling #4824), CEBPα (Santa Cruz Biotech sc-
166258), CEPBε (Santa Cruz Biotech sc-25570, PU.1 (Santa Cruz Biotech sc-352), p27 (Cell Signaling #3686)
and anti-β-Actin peroxidase (Sigma-Aldrich #A3854).
Murine studies: All experiments were approved by the Cleveland Clinic and Cincinnati Children’s Hospital
Medical Center Institutional Animal Care and Use Committees (IACUC). Cultured normal HSC, MLL-AF9 cells,
p53-null MLL-AF9 cells (THP1) cells or fresh patient bone marrow AML cells were transplanted by intravenous
injection into non-irradiated 6-8 week old NOD/SCID or NSG mice. Mice were anesthetized with isofluorane
before transplantation. Animals were checked daily and were euthanized by an IACUC approved method for
signs of distress. Bone marrow was analyzed by flow-cytometry, Giemsa-stain and Western blot. The
proportion of human hematopoietic cells in bone marrow was determined by positive staining with PC5-
conjugated anti-human CD45 mAb (BD) with isotype-matched immunoglobulin as a control in all experiments.
Livers and spleens were weighed and fixed. To anatomically localize THP-1cells in living mice, the substrate
D-Luciferin (15 mg/ml D-luciferin in sterile PBS (Promega), was injected IP and mice were imaged after 10
minutes mice using an IVIS-200 CCD camera imaging system (Xenogen, Alameda,CA).
Correlation of KI67 gene expression with GI50: Quality controlled raw data (Affymetrix CEL files, SOFT files)
from previously published experiments (GSE5846 (34)) were downloaded from Gene Expression Omnibus
(GEO) datasets (www.ncbi.nlm.nih.gov/geo). KI67 gene expression data in 6 leukemia cell lines (CCRF-CEM,
HL60, K562, MOLT4, RPMI8226, SR) was correlated with the decitabine concentration that produced 50%
growth inhibition (GI50) (data from Developmental Therapeutics Program of the NCI
[http://dtp.nci.nih.gov/index.html]). Scatter plots, Spearman and Pearson correlation coefficients were
generated using SAS statistical analysis software.
Equimolar decitabine or cytarabine in p53 wild-type MLL-AF9 cells. Identical concentrations of decitabine
and cytarabine (cytosine analogues metabolized through the same nucleotide pathways) were added to p53
wild-type MLL-AF9 AML cells (33). Decitabine 0.5 µM depleted DNMT1 (figure 1A) without causing significant
apoptosis (figure 1B) (annexin staining quantified by flow-cytometry 24 hours after decitabine or cytarabine
treatment). In contrast, an equimolar concentration of cytarabine caused substantial apoptosis (figure 1B).
Both decitabine and cytarabine 0.5 µM added on days 1 and 4 decreased AML cell proliferation (figure 1C).
Colony formation in methyl-cellulose is an assay for stem and progenitor cell activity. Both decitabine and
cytarabine substantially decreased colony formation by p53 wild-type MLL-AF9 cells (figure 1C). However, at
day 4, only decitabine treated MLL-AF9 cells displayed morphologic changes of monocyte differentiation
(increased cell size, decreased nuclear-cytoplasmic ratio, granulation and vacuolization of the cytoplasm)
(figure 1D). Cytarabine treated cells were small and disrupted, suggesting apoptosis and necrosis (figure 1D).
Both decitabine and cytarabine treatment increased expression of the monocyte marker CD14 (measured by
flow-cytometry on day 4), although the increase produced by decitabine was greater (median fluorescence
intensity 6.02 versus 4.09), quantified by flow-cytometry 96 hours after decitabine or cytarabine treatment)
(figure 1E). Neither drug increased expression of the granulocyte marker CD11b.
Differential effect on AML leukemia initiating cells and normal hematopoietic stem cells. Engraftment in
an immuno-compromised murine host is a functional assay for both normal HSC and leukemia initiating cells
(35;36). Normal CD34+ hematopoietic cells and p53 wild-type MLL-AF9 cells were treated with the identical
regimen of decitabine 0.5 µM (days 1 and day 5) and decitabine 0.2 µM (days 2 and day 6) (the objective was
to maximize in vitro exposure to decitabine but without reaching concentrations that produce measurable DNA
damage). On day 7, equal numbers (3 x 105 cells each) of viable normal and MLL-AF9 cells were combined
and transplanted into sub-lethally irradiated NOD/SCID recipient mice. The mice receiving the combination of
mock treated normal and mock treated MLL-AF9 cells required euthanasia by week 6 and demonstrated
extensive bone marrow engraftment with human leukemia cells (figure 2A, B, figure S1). Mice receiving the
combination of decitabine-treated normal and decitabine-treated MLL-AF9 cells remained healthy and were
sacrificed at week 13 (greater than twice the period of survival of the control group) (figure 2A, figure S1).
These mice demonstrated normal human hematopoietic cell engraftment, comparable to that seen in mice
receiving 4x106 normal human CD34+ cells without leukemia cells. Analysis of their BM showed no
morphologic or flow-cytometric evidence of leukemia cell engraftment (figure 2B, figure S1).
Equimolar decitabine or cytarabine in p53-null MLL-AF9 cells. The effects of equimolar decitabine and
cytarabine were then compared in p53-null MLL-AF9 AML cells (the THP1 AML cell line (37)). The
concentration of decitabine used depleted DNMT1 without causing significant apoptosis (Figure 3A, B). Unlike
p53 wild-type MLL-AF9 cells, only decitabine treatment impaired proliferation and decreased colony formation,
while cells treated with cytarabine continued to proliferate (figure 3C). Similar to p53 wild-type cells, only
decitabine treatment induced morphologic features of myeloid differentiation (decreased nuclear-cytoplasmic
ratio, increased cell size, granulation and vacuolization of the cytoplasm). Cytarabine treated cells retained
immature morphology (figure 3D). Decitabine treatment markedly increased expression of the granulocyte
marker CD11b, with slight effects on CD14 expression. Cytarabine treatment did not affect these differentiation
markers (figure 3E).
Differential regulation of apoptosis and differentiation protein expression by decitabine and cytarabine.
Key events associated with apoptosis and cell cycle exit are known; these include p53 serine-15
phosphorylation, upregulation of p53, and possibly upregulation of cyclin-dependent kinase inhibitor 1A
(p21/CDKN1A) and p16/CDKN2A (reviewed in (38)). Myeloid lineage-commitment and differentiation requires
lineage-specifying transcription factors such as CEBPα and PU.1 (reviewed in (39)). Late myeloid
differentiation and cell cycle exit is associated with upregulation of the key late transcription factor CEBPε (40-
43) and p27/CDKN1B (p27) (29-32), and possibly upregulation of p15/CDKN2B. The regulation of these
apoptosis and differentiation events by decitabine and cytarabine was examined in p53 wild-type and p53-null
In p53 wild-type MLL-AF9 cells, cytarabine and to a lesser extent decitabine increased p53 serine-15
phosphorylation, p53 and p21/CDKN1A levels (figure 4A). p16/CDKN2A protein was not detected in these
cells using two different antibodies (figure 4A), although p16 mRNA was detected (data not shown). With
regard to differentiation events, in p53 wild-type cells, CEBPα protein was decreased by both drugs, whereas
no change was detected in PU.1 levels. The most striking change was in CEBPε protein, which peaked at 72
hours after decitabine treatment, and p27/CDKN1B protein, which peaked 96 hours after decitabine treatment
(figure 4A). Cytarabine had minimal effects on the expression of these key late differentiation proteins (figure
4A). Despite use of two separate antibodies from different manufacturers, we were unable to detect
p15/CDKN2B protein. However, decitabine treatment did increase CDKN2B mRNA >2-fold measured by QRT-
PCR (data not shown).
Similar to p53 wild-type cells, the most striking change in p53-null THP1 cells was a decitabine-induced
increase in CEBPε and p27/CDKN1B protein levels (figure 4B). Cytarabine did not produce these effects
(figure 4B). p21/CDKN1A levels were also increased by decitabine but not by cytarabine. CEBPα levels
decreased with both decitabine and cytarabine, whereas PU.1 levels decreased only in decitabine treated
cells. Neither p15 nor p16 mRNA nor protein were detected in the THP1 cells (figure 4B, data not shown).
Sensitivity of leukemia cell lines to decitabine inversely correlates with the proliferative index. The
proliferation index of leukemia cells may predict sensitivity to decitabine, since decitabine is S-phase specific in
its mechanism of action. In 6 leukemia cell lines, the concentration of decitabine that produced 50% growth
inhibition (GI50) inversely correlated with the expression of KI67 (a proliferation marker expressed only in
cycling cells; KI67 expression is widely used in clinical pathology as an index of malignant cell proliferation)
(GI50 data from the Developmental Therapeutics Program of the NCI [http://dtp.nci.nih.gov/index.html]; gene
expression data from GEO Datasets GSE5846 (34)) (figure S2).
Better survival with non-cytotoxic decitabine than with cytotoxic cytarabine in murine xeno-transplant
models of p53-null human AML. The correlation between sensitivity of AML cells to decitabine and the
proliferation index emphasizes the importance of optimizing scheduling and administration, to increase and
distribute the windows of drug exposure and capture AML cells entering S-phase at different points in time
(figure S3). Since the objective is not high peak levels of drug to cause DNA damage and cytotoxicity, sub-
cutaneous (SC) administration could have advantages over intra-peritoneal (IP) or intravenous administration
by producing lower peak levels but longer half-life. Since such doses are non-cytotoxic (24), it is feasible to
administer drug more frequently, to create more windows of drug exposure (figure S3). To confirm that 0.2
mg/kg administered SC weekly depletes DNMT1 without causing cytotoxicity or severe cytopenia in vivo, non-
transplanted NSG mice (n=4) were treated 1-2X/week for 8 weeks. There was no treatment associated
cytopenia (figure S4A). DNMT1 was substantially depleted in bone marrow cells analyzed at sacrifice with no
measurable increase in apoptosis and a small increase in phospho-H2AX levels (figure S4B-D).
Weekly SC decitabine was then used to treat a xeno-transplant model of p53-null human MLL-AF9 AML. Non-
irradiated NSG mice were transplanted with 3x106 THP1 cells by tail vein injection. Starting at day 5 after
transplant, mice were treated with vehicle (PBS), cytarabine 75 mg/kg/day IP for five consecutive days (to
model conventional chemotherapy (44)), or decitabine 0.2 mg/kg SC 3X/week for 2 weeks then 2X/week for 2
weeks then 1X/week thereafter. Mice treated with decitabine had significantly longer median survival (>20%
increase) than cytarabine and vehicle treated mice (median survival 51, 45, and 42 days respectively, Log-
Rank p=0.0004) (figure 5A). In vivo luminescence imaging on day 28 of therapy demonstrated disseminated
disease in vehicle treated mice but disease concentrated in the region of the liver in decitabine and cytarabine
mice (figure 5B). This pattern was recapitulated at sacrifice, when vehicle treated mice demonstrated
disseminated tumor masses in the thoracic and abdominal cavities and subcutaneously, but disease was
concentrated in the livers of cytarabine and decitabine treated mice, with large numbers of leukocytic nodules
(figure 5C). Spleen weights and sizes (obtained at different time-points of euthanasia as per the Kaplan-Meir
plot) were comparable between the different treatment groups, although decreased to a non-statistically
significant extent in decitabine treated mice (figure 5D). The liver expresses high levels of cytidine deaminase,
the enzyme that rapidly metabolizes cytosine analogues, which could explain liver sanctuary for liver tropic
THP1 cells from the effects of cytarabine and decitabine.
Better survival with non-cytotoxic decitabine than with cytotoxic cytarabine in a murine xeno-
transplant model of refractory/relapsed human AML. To complement the above experiment in which an
AML cell line was used, a xeno-transplant model was established using fresh AML cells from a patient with
relapsed/refractory AML. These AML cells contained multiple chromosome abnormalities including a
t(8;18)(q22:q23) and t(11;13)(q21:q12). Non-irradiated NSG mice were transplanted with 1x106 patient cells by
tail vein injection. Starting at day 5 after transplant, mice were treated with vehicle (PBS), cytarabine 75
mg/kg/day IP for five consecutive days (44), or decitabine 0.2 mg/kg SC 3X/week for 2 weeks then 2X/week for
2 weeks then 1X/week thereafter. Mice treated with decitabine had significantly longer median survival (>100%
increase) than cytarabine or vehicle treated mice (median survival 113, 56, and 50 days respectively, Log-
Rank p<0.0001) (figure 6A). At euthanasia (at different time-points corresponding to the Kaplan-Meir plot), the
bone marrow of all mice was replaced by human leukemia cells (figure 6B, figure S5A, B); spleens of
decitabine treated mice were significantly decreased in size compared to cytarabine or vehicle treated mice
(figure 6C, figure S5C). Unlike the THP1 cells, these AML cells did not demonstrate liver tropism (livers were
not increased in size or weight at sacrifice).
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 (25-28;45). 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 (45). Therefore, baseline differentiation stage is a major determinant of the
cell fate response to decitabine treatment. Surface phenotype can be used to sort AML cell populations into
subsets. These subsets can then be xeno-transplanted into immunocompromised mice for evaluation of
leukemia-initiating efficiency. The earliest studies suggested that AML cells with leukemia-initiating capacity
had a surface phenotype resembling that of normal HSC (CD34+38-) (36;46). 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 AML cell population (46). Recently, it has been reported
that the antibodies used to sort for CD38+ may inhibit proliferation and might have technically influenced the
earliest studies (47). Accordingly, in a number of recent studies, AML initiating cells had a surface phenotype
suggesting lineage-commitment (CD34+38+, CLL-1+, CD71+, CD90 -, c-Kit -) (47-53). Cross-species barriers
are another major influence on the outcome of leukemia or cancer initiating cell assay experiments (54). With
use of more immuno-compromised mice, or mice which express human cytokines, AML initiating cell surface-
phenotypes are not stem cell restricted (54-56). Differentiation absolutely requires and is driven by lineage-
specifying transcription factors such as CEBPA. CEBPA is expressed at high levels with lineage-commitment.
AML cells, including CD34+ and CD34+38- subsets express high levels of CEBPA, but relatively low levels of
the key late differentiation driver CEBPE (supporting 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, 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 also 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 intravenous
administration is in the order of minutes, compared to many hours in vitro (57;58). An obvious mechanism for
treatment failure therefore is that some or many AML cells may complete S-phase while decitabine is absent
from the system (most of the time). Decitabine was originally developed for cytotoxic therapy (59). Therefore,
doses to treat AML were escalated to maximum tolerated levels (up to 80 mg/kg infused over 36-44 hours) in
traditional phase 1 studies (57), followed by many weeks without treatment to allow for recovery from cytotoxic
side-effects. 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 (60). 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 (60;61).
The results here provide a biological rationale to continue this clinical trend of decreasing dose and
administering drug more frequently: very low drug levels are sufficient for non-cytotoxic DNMT1 depletion, and
the decrease in toxicity can be used to administer treatment frequently, to increase windows of drug exposure
and capture AML cells entering S-phase at different points in time (figure S3). That lower doses of SC
decitabine (0.2 mg/kg, ~5 mg/m2) are clinically active, with non-cytotoxic epigenetic and differentiation
modifying effects, and can be administered safely from 1-3 X/week, has been demonstrated in sickle cell
disease and β-thalassemia clinical trials (24).
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. Pharmacologic barriers to optimal clinical translation remain. However, these can potentially be
addressed through further pre-clinical and clinical investigation.
We gratefully acknowledge the following gifts: Mary Laughlin and Nick Greco at the Abraham J and Phyllis
Katz Cord Blood Foundation and Cleveland Cord Blood Center for cord blood samples; YS is supported by
NIH (1R01CA138858, U54HL090513) and Dept. of Defense (PR081404). KPN and YS are supported by Scott
Hamilton CARES Foundation.
Supplementary information is available at Leukemia's website
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