Loss of p53 Accelerates the Complications of Myelodysplastic Syndromes (MDS) In the NUP98-HOXD13 Transgenic Mouse Model

Abstract Abstract 2804 The myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis leading to blood cytopenias and a high rate of progression to acute myelogenous leukemia (AML) (Nimer, 2008). Recent studies implicated an important role for p53 in 5q−MDS, a subtype where haploinsufficiency of the RPS14 ribosomal protein drives the anemia that accompanies this disease (Ebert et al., 2008; Barlow et al., 2010 and Dutt et al., 2011). The elevated level of p53 activity apparently triggers the excessive apoptosis and the dysplastic morphology seen in the erythroid cells of the “5q-mice” that lack one copy of the chromosomal region syntenic to the human 5q region that contains the RPS14 gene (Barlow et al., 2010). Thus, the cytopenias in some patients with MDS may be the result of defective ribosomal biosynthesis, leading to activation of p53 and excessive apoptosis. The nucleoporin gene NUP98 is fused to a number of different genes including HOXD13 by chromosomal translocations that are found in patients with MDS, AML and CML, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia (Lin et al., 2005). We obtained these mice and analyzed the hematopoietic stem cells (HSC) and the erythroid compartment and found significantly decreased rps14 mRNA expression in the NHD13+ CD71+Ter119+ cells compared to the WT controls. Furthermore, flow cytometry analysis revealed increased intracellular p53 levels in the NHD13+ Lin−Sca-1+ c-Kit+ (LSK) and CD71+Ter119+ cells. These data suggest that defective rps14 ribosomal protein production and an increased p53 level may contribute to the anemic phenotype in NHD13+ Tg mice. To examine whether inhibition of p53 function can improve the cytopenias of NHD13+ Tg mice, mice were injected with Pifithrin-α (a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription, 2 mg/kg body weight) daily for five weeks. We observed partial rescue of the myeloid and lymphoid lineage differentiation defects, with no improvement in the hemoglobin level. To further investigate whether the presence or absence of p53 affects the MDS or AML phase of NHD13 driven disease, we generated NHD13+p53+/− and NHD13+p53−/− mice. Deletion of one allele of p53 rescues the myeloid progenitor cell compartment, while deletion of both alleles reversed the deficit in both the LSK and the MPP populations that was observed in the NHD13+ Tg mice. The clinically healthy NHD13+p53−/− mice (aged 3 to 5 months) and the NHD13+p53+/− mice (aged from 3 to 7 months) both had more severe leukopenia and anemia than the NHD13+ Tg mice or the WT controls (aged 4 to 7 months). 60% of the NHD13+p53−/− mice and the NHD13+p53+/− mice developed MDS with a median survival of 138 d and 190 d respectively; in contrast 30% of the NHD13+ Tg mice had MDS with a median survival of 236 d. These data indicate that the relative or absolute lack of p53 hastens the development of MDS, and shortens the median survival. Lack of one or two p53 alleles significantly accelerated the development of AML, which in NHD13+p53+/− mice resulting in a median survival of 278 d. However, AML in the NHD13+p53−/− mice is more undifferentiated with a median survival of 133 d, and the NHD13+ Tg mice showed AML with a median survival of 324 d. Taken together, these data demonstrated that the chronic loss one allele or two alleles of p53 does not rescue the MDS phenotype induced by NHD13 fusion gene. Rather it accelerates the development of NHD13 driven MDS and leukemia. Our studies suggest that targeting p53 transiently may temporally improve hematopoiesis in MDS, but over the long term has detrimental effects on hematopoiesis. Disclosures: No relevant conflicts of interest to declare

Identification of Key Cellular Regulators That Interact with Id1 To Control Hematopoietic Stem Cell Behavior

Abstract The ability of hematopoietic stem cells to tightly regulate the transition from relative quiescence and self-renewal to the transiently amplifying, differentiating progenitor fate is critical for HSC homeostasis as well as their regenerative capacity. We have recently described the diminished frequency and rapid exhaustion of HSC self-renewal capacity in the absence of the dominant negative helix-loop-helix molecule Id1. Furthermore, Id1 null HSCs have an increased rate of cycling, coupled with accelerated myeloid commitment both in vivo and in vitro. This is reflected in the elevated expression of myelo-erythroid transcription factors (c/EBPalpha and GATA1) within the Lin−c-kit+Sca-1+ population - “myeloid priming”. The major targets of Id1 mediated transcriptional repression are the ubiquitous E protein E2A as well as Ets transcription factors (Ets1 and Ets2). We hypothesized that the unrestrained activity of these and/or other targets of Id1 transcriptional repression leads to premature HSC commitment in Id1 null animals. Indeed, we show that HSC differentiation in culture can be delayed by transduction of E2A directed shRNA specifically in Id1 null, but not in wild-type Id1 expressing cells. This indicates an abnormal E2A activity in Id1 null HSCs that could be responsible for their increased differentiation status. To further define the transcriptional deregulation in Id1 null HSCs, we have used the Affymetrix microarray technology. We observed ~3 fold increased expression of the CDK inhibitor p21 in freshly isolated Id1 null HSCs and have confirmed this result by multiple independent qPCR measurements. The transcriptional induction of p21 by E2A as well as its repression by Id1 have been well established. Therefore, the observed p21 induction could be explained by the elevated level of E2A activity in HSCs in the absence of Id1 expression. To explore the functional significance of Id1 mediated p21 regulation in HSCs, we have generated p21/Id1 double knockout animals. Surprisingly, despite its reported function in restricting the cell cycle entry of normal HSCs, we show that in the context of Id1 loss, p21 expression is required for the accelerated HSC cycling, and unlike Id1 single null HSCs, p21/Id1 double knockout HSCs do not show accelerated myeloid differentiation in culture. Therefore, we propose that Id1 actively represses E2A activity in HSCs, as well as the induction of p21, which could be an important component of the HSC commitment program. Further studies will be presented defining the in vivo relevance of the Id1/p21 genetic interaction for HSC growth and differentiation

Id1 restrains myeloid commitment, maintaining the self-renewal capacity of hematopoietic stem cells

Appropriate hematopoietic stem cell (HSC) self-renewal reflects the tight regulation of cell cycle entry and lineage commitment. Here, we show that Id1, a dominant-negative regulator of E protein transcription factors, maintains HSC self-renewal by preserving the undifferentiated state. Id1-deficient HSCs show increased cell cycling, by BrdU incorporation in vivo , but fail to efficiently self-renew, leading to low steady-state HSC numbers and premature exhaustion in serial bone marrow transplant assays. The increased cycling reflects the perturbed differentiation process, because Id1 null HSCs more readily commit to myeloid differentiation, with inappropriate expression of myeloerythroid-specific genes. Thus, Id1 appears to regulate the fate of HSCs by acting as a true inhibitor of differentiation

Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators

The AML/RUNX family of transcription factors plays important roles in hematopoiesis, neurogenesis, bone development, and segmentation in vertebrate embryos. The aim of this study was to isolate runt-related genes in a genetically and embryologically exploitable system, the zebrafish, and characterize their function during hematopoietic development. Two runt-related genes were isolated by degenerate PCR and standard library screening, and a radiation hybrid panel, T51 RH, was used to resolve their chromosomal localization. In situ hybridization demonstrated their expression whereas their transcriptional activity was assessed using an AML1-responsive reporter gene in the MLA 144 T-cell line. We isolated the zebrafish runxa and runxb cDNAs, which encode proteins highly homologous to the human and murine Runx1 (AML1) and Runx3 (AML2) proteins. In contrast to a recent report, we detected runxa expression in both hematopoietic and neural tissues of the developing zebrafish. runxa transcripts first appear during segmentation in bilateral mesodermal cells that coexpress one of the earliest blood and endothelial cell markers, scl/tal-1. By 24 hours postfertilization (hpf), runxa transcripts are seen in the ventral wall of the dorsal aorta. Hematopoietic runxa expression is lost in cloche mutants, which are defective in blood and endothelial cell formation. runxb transcripts are seen in nonhematopoietic domains. Both Runxa and Runxb transactivate an AML1-responsive human promoter in hematopoietic cells. Genomic localization studies demonstrate that runxa is located on linkage group 1 (LG1), and the runxb gene is located on LG13. Our gene expression analysis strongly suggests that both the functional and spatial aorta-gonad-mesonephros (AGM) region has been conserved throughout evolution. Our runxa spatiotemporal expression data shed light on the role of vertebrate Runx1/AML1 in primitive vs definitive hematopoietic development

Prognostic significance of minimal residual disease detection and PML/RAR-α isoform type: long-term follow-up in acute promyelocytic leukemia

Abstract The t(15;17) translocation in acute promyelocytic leukemia (APL) yields a PML/RAR-α fusion messenger RNA species that can be detected by reverse transcription–polymerase chain reaction (RT-PCR) amplification. Breakpoints within intron 3 of PML produce a short PML/RAR-α isoform, whereas breakpoints within intron 6 result in a longer form. Using RT-PCR, serial evaluations were performed on the bone marrow of 82 patients with APL (median follow-up, > 63 months) who received retinoic acid (RA) induction followed by postremission treatment with chemotherapy, RA, and biologic agents. Sixty-four patients attained a clinical complete remission and had at least 2 RT-PCR assays performed after completing therapy. Forty of 47 patients (85%) with newly diagnosed APL who were induced using RA had residual disease detectable by RT-PCR before additional therapy. After 3 cycles of consolidation therapy, residual disease was found in only 4 of 40 evaluable patients (10%). Among newly diagnosed patients who had 2 or more negative RT-PCR assays, only 3 of 41 (7%) had a relapse, whereas all 4 patients (100%) who had 2 or more positive results had a relapse. Among 63 newly diagnosed patients, those who expressed the short isoform appeared to have shorter disease-free and overall survival durations than patients who expressed the long isoform. These data indicate that 2 or more negative RT-PCR assays on bone marrow, performed at least 1 month apart after completing therapy, are strongly associated with long-term remissions. Conversely, a confirmed positive test is highly predictive of relapse

Loss of p53 accelerates the complications of myelodysplastic syndrome in a NUP98-HOXD13–driven mouse model

The nucleoporin gene NUP98 is fused to several genes including HOXD13 in patients with myelodysplastic syndromes (MDS), acute myeloid leukemia, and chronic myeloid leukemia, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia. Here we show that short-term treatment with the p53 inhibitor Pifithrin-α partially and transiently rescued the myeloid and lymphoid abnormalities found in NHD13 + Tg mice, with no improvement in the anemia, while the genetic deletion of 2 alleles of p53 rescued both the myeloid progenitor cell and long-term hematopoietic stem cell compartments. Nonetheless, loss of one or both alleles of p53 did not rescue the MDS phenotype, but instead exacerbated the MDS phenotype and accelerated the development of acute myeloid leukemia. Our studies suggest that while targeting p53 may transiently improve hematopoiesis in MDS, over the long-term, it has detrimental effects, raising caution about abrogating its function to treat the cytopenias that accompany this disease

The Acetylation of AML1-ETO Is Required for Leukemogenesis

Abstract Abstract 1588 Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare

PRMT4 Blocks Myeloid Differentiation by Assembling a Methyl-RUNX1-Dependent Repressor Complex

Defining the role of epigenetic regulators in hematopoiesis has become critically important, because recurrent mutations or aberrant expression of these genes has been identified in both myeloid and lymphoid hematological malignancies. We found that PRMT4, a type I arginine methyltransferase whose function in normal and malignant hematopoiesis is unknown, is overexpressed in acute myelogenous leukemia patient samples. Overexpression of PRMT4 blocks the myeloid differentiation of human stem/progenitor cells (HSPCs), whereas its knockdown is sufficient to induce myeloid differentiation of HSPCs. We demonstrated that PRMT4 represses the expression of miR-223 in HSPCs via the methylation of RUNX1, which triggers the assembly of a multiprotein repressor complex that includes DPF2. As part of the feedback loop, PRMT4 expression is repressed posttranscriptionally by miR-223. Depletion of PRMT4 results in differentiation of myeloid leukemia cells in vitro and their decreased proliferation in vivo. Thus, targeting PRMT4 holds potential as a novel therapy for acute myelogenous leukemia

The Leukemogenicity of AML1-ETO Is Dependent on Site-Specific Lysine Acetylation

The chromosomal translocations found in acute myelogenous leukemia (AML) generate oncogenic fusion transcription factors with aberrant transcriptional regulatory properties. Although therapeutic targeting of most leukemia fusion proteins remains elusive, the posttranslational modifications that control their function could be targetable. We found that AML1-ETO, the fusion protein generated by the t(8;21) translocation, is acetylated by the transcriptional coactivator p300 in leukemia cells isolated from t(8;21) AML patients, and that this acetylation is essential for its self-renewal–promoting effects in human cord blood CD34 + cells and its leukemogenicity in mouse models. Inhibition of p300 abrogates the acetylation of AML1-ETO and impairs its ability to promote leukemic transformation. Thus, lysine acetyltransferases represent a potential therapeutic target in AML