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

Abstract

Abstract 2077Poster Board II-54Conventional 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. Disclosures:Off Label Use: Decitabine, to treat myelodysplastic syndrome using a novel dose and schedule. Advani:Cephalon: Research Funding. Saunthararajah:HemaQuest: Consultancy.