386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

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Purpose. The positive lenalidomide experience in lower risk myelodysplastic syndrome (MDS) with deletion 5q and red blood cells transfusion dependence resulted in its broad use in MDS. The study aim was to define the prognosis in MDS and deletion 5q with or without other cytogenetic abnormalities. Patients and Methods. Patients with MDS (<20% blasts) and evaluable cytogenetic studies (1966-present) were reviewed. Outcome of patients with deletion 5q or with loss of chromosome 5 (monosomy 5) was evaluated by the presence or absence of additional chromosomal abnormalities, overall, and in “lower risk” MDS. Results. Of 2743 patients analyzed, 287 (10%) had deletion 5q, and 216 (8%) had monosomy 5 abnormalities. The median survival was 9 months with deletion 5q, 6 months with monosomy 5, and 17 months without a chromosome 5 abnormality. Considering patients with deletion 5q, the median survival was 33 months with deletion 5q alone, 17 months with deletion 5q and one additional abnormality, but only 6–12 months with deletion 5q and ≥ 2 abnormalities or chromosome 7 abnormality. Only 93 patients (3.4% of total) had lower risk MDS (International Prognostic Scoring System low or intermediate 1) and deletion 5q; their median survival 29 months (2-year survival 60%), and ranged from 13 to 41 months depending on the presence or absence of additional chromosomal abnormalities. Among 58 patients with lower risk MDS deletion 5q with identical entry criteria to the original lenalidomide study, the median survival was 44 months and the 2-year survival rate 80%. Among 198 patients with MDS with deletion 5q and < 10% blasts (a subset now often offered lenalidomide) the median survival was 12 months. Monosomy 5 was significantly more frequently associated with advanced MDS and with other chromosomal abnormalities. Considering only patients with IPSS intermediate 2 or high risk, the survival was better with deletion 5q versus monosomy 5 (medians 7 versus 6 months; p = 0.005). Conclusions. The prognosis of MDS with chromosome 5 abnormalities is heterogeneous. This study provides baseline expectations of outcome in different MDS subsets and deletion 5q, which could be used for comparative purposes with the lenalidomide experience.

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