ALBERT

Description: High salinity adversely affects crop production. Pyruvic acid is the precursor of abscisic acid (ABA) and other chemicals that are synthesized in chloroplast, some of which are involved in the response to salt...

Description: The mixed-lineage leukemia (MLL) H3K4 methyltransferase protein, and the heterodimeric RUNX1/CBFβ transcription factor complex, are critical for definitive and adult hematopoiesis, and both are frequently targeted in human acute leukemia. We identified a physical and functional interaction between RUNX1 (AML1) and MLL and show that both are required to maintain the histone lysine 4 trimethyl mark (H3K4me3) at 2 critical regulatory regions of the AML1 target gene PU.1. Similar to CBFβ, we show that MLL binds to AML1 abrogating its proteasome-dependent degradation. Furthermore, a subset of previously uncharacterized frame-shift and missense mutations at the N terminus of AML1, found in MDS and AML patients, impairs its interaction with MLL, resulting in loss of the H3K4me3 mark within PU.1 regulatory regions, and decreased PU.1 expression. The interaction between MLL and AML1 provides a mechanism for the sequence-specific binding of MLL to DNA, and identifies RUNX1 target genes as potential effectors of MLL function.

Description: One mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is through partial tandem duplication (MLL-PTD); however, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state because of increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT-derived phenotypic short-term (ST)–HSCs/multipotent progenitors and granulocyte/macrophage progenitors have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. Thus, the Mll-PTD aberrantly alters HSPCs, enhances self-renewal, causes lineage bias, and blocks myeloid differentiation. These findings provide a framework by which we can ascertain the underlying pathogenic role of MLL-PTD in the clonal evolution of human leukemia, which should facilitate improved therapies and patient outcomes.

Description: Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we've recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn't develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

Description: Aberrant transcriptional programs play a critical role in the development of acute myeloid leukemias (AMLs). Although persistent over-expression of MEIS1 and HOXA9 has been shown to be essential for the initiation and maintenance of MLL-associated leukemia, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion-driven MEIS/HOX pathway, dictate the development of MLL leukemia. Considering that AMLs with MLL translocation are typically associated with the monocytic lineage (FAB M4 and M5), we explored the potential role of the monocytic lineage-specific transcriptional program in MLL leukemia. Using 97 genome-wide expression profiles of human MLL leukemias, we constructed an MLL distinctive transcriptional regulatory network. In addition to well-known transcriptional factors in leukemia development such as MEIS1 and HOXA family genes, we identified a highly active monocyte-specific gene signature that includes transcription factor PU.1. In our effort to determine the functional role of PU.1 in MLL leukemia, we found that lower PU.1 expression significantly delayed the onset of MLL- AF9 induced leukemia in primary bone marrow transplantation assay. MLL leukemia failed to maintain in vivo upon induced deletion of the PU.1 gene. To examine the clinical relevance of the PU.1 in AML patients, we further performed multivariate Cox proportional-hazards regression analysis in four published datasets of patients with AML, for whom gene expression and time-to-event data were available. We found that a PU.1-regulated 40-gene signature showed profound concordance with prognosis in segregating high-risk and low-risk AML patients. When specific subgroups of AMLs were examined, the PU.1 expression signature could predict patient outcome for MLL patients, but not in other major AMLs, such as t(8;21), t(15;17) and inv(16). We further explored the molecular mechanisms underlying the critical role of the PU.1 program in MLL leukemia. Functional annotation of this PU.1 expression signature identified the MEIS/HOX pathway (MEIS1, FLT3, KIT), as well as key genes in the inflammatory response (AIF1, NF-KB1 and CD180). We showed that PU.1 is required to maintain high expression of Meis1 and Pbx3 and also important downstream genes in the MEIS/HOX pathway that includes known MEIS/HOX targets c-Kit and Flt3. Using ChIP-sequencing, we demonstrated that PU.1 interacts with the MEIS/HOX regulatory program through co-binding with MEIS1 at the target genomic regions in a MLL-ENL cell line. In our effort to determine the role of PU.1-controlled inflammatory response genes, we found that the growth inhibition in PU.1 knockdown MLL leukemic cells was partially rescued by addition of the monocytic inflammatory cytokine AIF1. AIF1 provides an anti-apoptotic effect through activation of the NF-ƒÛB pathway and additional known apoptosis regulators. Interestingly, AML patients with higher expression of both AIF1 and MEIS1 had a significantly shorter overall survival time than those with lower expression of both genes. Patients with high expression of either MEIS1 or AIF1 had medium survival possibilities. Notably, the prognostic value of AIF1 and MEIS1 remained in those with monocytic AMLs (P=0.00079), but not in the non- monocytic group of patients (p=0.105). Collectively, these results strongly suggest that the monocyte-specific inflammatory cytokine AIF1 is an MEIS/HOX independent essential regulator in monocytic AMLs such as MLL leukemia. Loss of function PU.1 is leukemogenic in mouse models. Suppression of PU.1 activity is also required for the development of human myelocytic M2/M3 leukemia. Here we reveal a converse role for PU.1 as an essential positive regulator in the development of MLL myeloid leukemia, mostly M4/M5 monocytic AMLs. Our study demonstrats that the monocyte-specific PU.1-driven transcriptional program independently contributes to the development of myeloid MLL leukemia, in parallel with the MLL fusion pathway. PU.1 and downstream macrophage specific inflammatory cytokine AIF1 have important prognostic value and may serve as novel therapeutic targets for MLL leukemias. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 674 The interplay between genetic and epigenetic regulators is a fundamental mechanism to ensure gene regulation and cell fate decision. Hematopoiesis and leukemia are excellent systems in which to study this process. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcription factor RUNX1 (AML1)/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. PU.1 is a critical downstream target gene for AML1 in adult hematopoiesis. AML1 regulated PU.1 through 3 AML1 binding sites in the PU.1 URE region (1). AML1 is responsible for the H3K4me3 mark at PU.1 URE and promoter region. AML1-ETO represses PU.1 expression through PU.1 URE region (2). Dysregulation of PU.1 levels cause leukemia in both mouse and human. PU.1 is absolutely required for the normal development of monocytic and B cell lineages, in which the core binding factor (CBF) fusions (CBFβ-SMMHC or TEL-AML1) or MLL fusions (MLL-AF9 or MLL-AF4) have been high frequently identified in human acute leukemias. These suggested that AML1/CBFβ and MLL might regulate PU.1 expression and AML1/CBFβ or MLL fusions might dysregulate PU.1 expression at genetic and epigenetic levels and eventually develop similar leukemias. We found that the AML1-ETO/CBFβ complex interacts with MLL in a similar manner as AML1/CBFβ (3), while the AML1/CBFβ-SMMHC complex interacts with MLL more strongly. Surprisingly, the CBFβ-SMMHC by itself interacts with MLL through the junction region of the fusion protein. The interactions with MLL by these fusion proteins complexes, AML1-ETO/CBFβ and AML1/CBFβ-SMMHC, correlate with their ability to maintain H3K4me3 levels at PU.1 URE and promoter regions in 416B cell lines stable expressing AML1-ETO or CBFβ-SMMHC. Taken together, our data indicate that AML1-ETO/CBFβ complex preserves the interaction with MLL, while the AML1/CBFβ-SMMHC complex enhances it. This suggests that these two leukemogenic fusions downregulate PU.1 expression through different epigenetic mechanisms in the presence of H3K4me3 mark at PU.1 regulatory region. The effects of these differential interactions on the H3K4me3 mark maintenance and on target gene expression, particularly PU.1, may be critical for the aberrant regulation that underlies the etiology of M2 and M4Eo acute myelogenous leukemia (AML). 1. Huang G, et al. Nat. Genet. 2008; 40: 51-60 2. Zhang P, et al. Blood 2008; 112: 594. 3. Huang G, et al. Blood 2008; 112: 282.

Description: Abstract 3501 Rearrangements of the Mixed-Lineage Leukemia (MLL) gene occur in a variety of aggressive human leukemias. The most common ones are balanced translocations in which the genomic sequences encoding the N-terminal portion of MLL are fused to sequences encoding the C-terminus of another translocation partner in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). Another mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and AML, but rarely seen in ALL, is partial tandem duplication (MLL-PTD). The MLL–PTD was first identified in de novo AML with a normal karyotype or trisomy 11. Cloning of this region revealed partial duplications within the 5′ region of the MLL gene. These duplications consist of an in-frame repetition of MLL exons in a 5′–3′ direction and produce an elongated protein. The incidence of MLL–PTD was 8% in unselected adult and childhood AML and 5% in MDS. However, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Mll-PTD knock-in mouse model, its expression is regulated by endogenous promoter, has been generated to study the function of Mll-PTD in vitro and in vivo and to identify its downstream targets. This mouse model provides a powerful genetic tool to identify disruptions in normal cellular regulation as a result of this mutation, as well as a model to characterize the contribution of the Mll-PTD in leukemogenesis. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state due to increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT–derived phenotypic short-term (ST)-HSCs/multipotent progenitors (MPPs) and granulocyte/macrophage progenitors (GMPs) have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. In conclusion, the MllPTD/WT mouse model provides unique genetic and biochemical tool to identify new targets and pathways responsible for the altered differentiation/repopulating properties, self-renewal activity, lineage bias and myeloid differentiation blockade relevant to MLL-PTD MDS and AML. This model should also help us to understand the underlying mechanism(s) for each of the phenotypes we found in this study and facilitate improved therapies and patient outcomes in the future. Disclosures: No relevant conflicts of interest to declare.