Non-p53 Dependent, Leukemia Initiating-Cell Selective, Therapy.

Abstract 2077

Poster Board II-54

Conventional drug therapy for AML is limited by toxic effects on normal hematopoietic stem cells (nHSC), and dependence on p53/apoptosis pathways that are impaired in malignancy. In hematopoiesis, key transcription factors (TF) determine cell-fate. Here, a difference in nHSC versus leukemia initiating-cell (LIC) TF expression is used to overcome the above limitations.

The DNA methylating enzyme DNA methyl-transferase 1 (DNMT1) is also a component of multi-protein histone methyl-transferase complexes. Accordingly, shRNA mediated depletion of DNMT1 in hematopoietic cells hypomethylated DNA and decreased global H3K27 and H3K9 trimethylation (histone marks associated with transcription repression) by >70%. These epigenetic modifications were reproduced using a clinically relevant method: the cytosine analogue decitabine, added to normal human CD34+ hematopoietic precursor cells at 0.2–0.5uM 2–3X/week, depleted DNMT1, H3K27 and H3K9 trimethylation by >70% and significantly hypomethylated DNA (Illumina CpG Microarray). These decitabine levels did not cause measurable DNA damage (H2AX phosphorylation and Fast Micromethod) or apoptosis (Annexin staining and caspase 3 activity). Therefore, at low levels, decitabine can produce broad chromatin changes that increase TF access to target genes, without causing measurable DNA damage or apoptosis.

The gene-expression/cell-fate consequences of opening chromatin with decitabine likely depend on the pre-existing TF expression pattern. HOXB4 (stem cell TF), CEBPa (lineage-specifying TF), and CEBPe (late differentiation TF) levels were measured by RQ-PCR in CD34+ cells from AML (n=3) versus normal bone marrow (n=3). AML CD34+ expressed >50-fold higher CEBPa, but HOXB4 and CEBPe levels comparable to normal CD34+ cells, a pattern confirmed in microarray gene expression analysis (CD34+ and myeloblasts, AML n=321, normal n=51 (GEO)). Repression of late differentiation TF likely involves chromatin-modification, regardless of underlying cause. Therefore, depleting DNMT1 to open chromatin in AML cells expressing high lineage-specifying TF could resume differentiation and terminate AML self-renewal, while nHSC, with high stem cell TF and little lineage-specifying TF, should continue to self-renew. nHSC and human MLL-AF9 AML cells were treated identically with decitabine for 7 days, then 300,000 each viable MLL-AF9 and nHSC were combined and transplanted into NSG mice (n = 8). Mice that received PBS treated cells died by week 5 (>90% human myeloblasts in bone marrow). Mice that received decitabine treated cells remained healthy until sacrifice for analysis at week 12 (log-rank p = 0.02, no detectable leukemia, >80% normal human hematopoietic cell marrow engraftment). Direct treatment of mice with established MLL-AF9 leukemia with very low dose decitabine 1mg/m2 3X/week extended survival by >20% (log-rank p = 0.04).

Decitabine 0.5uM 2X/week induced morphologic differentiation, but not early apoptosis, in primary patient samples (n=15) and leukemia cell-lines (n=4). Cell-cycle exit by differentiation versus apoptosis may utilize different cyclin dependent kinase inhibitors (CDKN). The THP1 AML cell line contains a homozygous frame-shift mutation in TP53 (p.R174fs*3) and no detectable p53 RNA/protein. THP1 cells were treated with equimolar Ara-C or decitabine. Ara-C weakly upregulated CDKN1A (p21) but not CDKN2B (p15), and produced a transient decrease in cell-counts (D3-5) with recovery and growth similar to control by D7. Decitabine strongly upregulated p15, weakly upregulated p21, and produced gradual but complete and durable abrogation of cell growth by D7.

A 66y patient with transfusion dependent RCMD with 5q-, 15q- and severe comorbidities was treated with metronomic (instead of cycled) very low dose SQ decitabine (0.2mg/kg [7.5mg/m2] 2X/week) to avoid cytotoxicity and sustain differentiation modification. Platelets increased by week 4, hematologic remission occurred by week 8 and cytogenetic remission by week 14 (without significant side-effects).

Rationalizing dose and schedule of decitabine exploits a difference in nHSC and LIC TF expression to selectively terminate LIC self-renewal by a non-p53 dependent differentiation pathway. This approach, distinct from conventional apoptosis-based therapy, could have a very favorable safety profile, with efficacy in MDS/AML with complex cytogenetic abnormalities.