RUNX1 Isoforms Research Articles

CD56 and RUNX1 isoforms in AML prognosis and their therapeutic potential

Neural cell adhesion molecule (NCAM/CD56) expression in acute myeloid leukemia (AML) has been associated with extramedullary leukemia, multidrug resistance, shorter remission and survival. Recently, Bloomfield et al. published a succinct review of issues surrounding the AML prognostication and current therapeutics. However, we want to reiterate the prognostic value and therapeutic potential of CD56 that is frequently expressed in AML as was also reported by their own group earlier. In addition, novel RUNX1 isoforms contribute in controlling CD56 expression in AML cells. Anti-CD56 antibody therapy deserves exploration as an arsenal against AML patients expressing CD56. Relevantly, targeting RNA splicing machinery or RUNX1 isoform-specific siRNA may also become part of future therapeutic strategies for AML with CD56 overexpression.

The Roles of RUNX1 in Human Hematopoiesis and Megakaryopoiesis Revealed By Genome-Targeted Human iPSCs and an Improved Hematopoietic Differentiation Model

The RUNX1 gene (also called AML1), one of the most mutated genes in acute myeloid leukemia (AML), was first identified decades ago that encodes a key regulatory transcriptional factor. Numerous studies using mouse and zebrafish models show that RUNX1 is essential for definitive hematopoiesis. In mice, its homozygous knock-out (KO) in hematopoietic stem/progenitor cells also causes defects in lymphoid and megakaryocytic (MK) development. However, heterozygous Runx1 gene mutations in laboratory mouse and zebrafish had little effects on development of hematopoietic stem/progenitor cells (HSPCs) or the MK cell lineage. In contrast, heterozygous germline mutations in RUNX1 were found in patients with familial platelet disorder (FPD) with predisposition to AML and MDS. The mechanisms underlying the observed differences between humans and small animal models remain unclear.In the past decade, we and others have utilized human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate human hematopoiesis including the roles of the RUNX1 gene. Using a feeder-free culture system, we generated human CD34+CD45+ HSPCs cells from human ESCs and iPSCs around 11-14 days after embryoid body (EB) formation. The CD34+CD45+ HSPCs were capable to form multiple types of hematopoietic cells such as myeloid, erythroid, and polyploid MK cells (Connelly et al., 2014; Liu et al., 2015). We also reported that human iPSCs derived from FPD patients containing a heterozygous RUNX1 mutation were defective in MK formation, and targeted correction of the mutated RUNX1 allele by genome editing restored the MK potential (Connelly et al., 2014).Since then, we have extended our studies by precise genomic targeting in human wildtype iPSCs to ablate exon 5 that is common in all 3 isoforms of the RUNX1 gene, or exon 1B that is unique to the RUNX1c isoform. Bi-allelic KO of RUNX1 at exon 5 completely abolished the formation of hematopoietic cells at days 11-14 after EB formation. Complete disruption of exon 1B showed little effect, indicating that the RUNX1c isoform is dispensable for definitive hematopoiesis in the presence of the RUNX1a and RUNX1b isoforms transcribed from the downstream P2 promoter. Detailed analysis of EBs at days 6-8 revealed that bi-allelic RUNX1 KO at exon 5 (ablating all 3 isoforms) did not affect the formation of CD34+/CD31+/CD144+ endothelial-like cells. However, the endothelial-to-hematopoietic transition (EHT) was completely blocked and no CD45+ hematopoietic cells emerged from the EHT culture supplemented with hematopoietic cytokines.To elucidate the functions of different RUNX1 isoforms in early steps of human hematopoiesis, we adapted the EHT culture system that uses CD34+ cells isolated from earlier stages of EB formation (day 6-8), before definite hematopoiesis was observable (after day 8, Bai et al., 2015). Two different iPSCs clones with homozygous or bi-allelic RUNX1 KO at exon 5 both failed to form CD45+ hematopoietic cells after EHT culture. Because constitutive transgene expression of RUNX1b or RUNX1c (but not RUNX1a) cDNA in human iPSCs inhibits hematopoietic differentiation, we transduced the day 6 EB cells at the beginning of the EHT culture with RUNX1-expressing vectors. Lentiviral vectors constitutively express RUNX1b or RUNX1c (but not RUNX1a) cDNA partially restored the EHT and hematopoietic (CD45+) cell formation after 4-5 days in EHT culture. We further used a lentiviral vector in which RUNX1c (as an ER fusion protein) can be conditionally activated by 4-HT induction. Induction starting at day 4 and lasting for 3 days rendered the maximal effect of hematopoietic cell formation in the EHT culture using CD34+ cells isolated at day 6 of EB formation.Our data corroborate with limited in vivo data using human fetal tissues on the possible roles of RUNX1 in definitive hematopoiesis. At present we are analyzing iPSC clones with mono-allelic disruption of exon 5 in a wild type iPSC line, comparing to iPSCs derived from FPD patients. The current study of using isogenic human iPSCs will help to understand the roles of RUNX1 in human hematopoiesis and megakaryopoiesis, and offer an amenable system to study the RUNX1 gene functions and downstream target genes. DisclosuresNo relevant conflicts of interest to declare.

An Intronic Suppressor Element Regulates RUNX1 Alternative Polyadenylation

Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types.Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways.To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions.Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. DisclosuresNo relevant conflicts of interest to declare.

Runx1 exon 6–related alternative splicing isoforms differentially regulate hematopoiesis in mice

AbstractRUNX1 is an important transcription factor for hematopoiesis. There are multiple alternatively spliced isoforms of RUNX1. The best known isoforms are RUNX1a from use of exon 7A and RUNX1b and c from use of exon 7B. RUNX1a has unique functions due to its lack of C-terminal regions common to RUNX1b and c. Here, we report that the ortholog of human RUNX1awas only found in primates. Furthermore, we characterized 3 Runx1 isoforms generated by exon 6 alternative splicing. Runx1bEx6−(Runx1bwithout exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the full-length Runx1b (Runx1bEx6+). They also facilitated the transactivation of Runx1bEx6+. To gain insight into in vivo functions, we analyzed a knock-in (KI) mouse model that lacks isoforms Runx1b/cEx6−and Runx1bEx6e. KI mice had significantly fewer lineage-Sca1+c-Kit+cells, short-term hematopoietic stem cells (HSCs) and multipotent progenitors than controls. In vivo competitive repopulation assays demonstrated a sevenfold difference of functional HSCs between wild-type and KI mice. Together, our results show that Runx1 isoforms involving exon 6 support high self-renewal capacity in vitro, and their loss results in reduction of the HSC pool in vivo, which underscore the importance of fine-tuning RNA splicing in hematopoiesis.

T‐cell receptor signaling induces proximal Runx1 transactivation via a calcineurin–NFAT pathway

Runx1 transcription factor is a key player in the development and function of T cells. Runx1 transcripts consist of two closely related isoforms (proximal and distal Runx1) whose expressions are regulated by different promoters. Which Runx1 isoform is expressed appears to be tightly regulated. The regulatory mechanism for differential transcription is, however, not fully understood. In this study, we investigated the regulation of the proximal Runx1 promoter in T cells. We showed that proximal Runx1 was expressed at a low level in naïve T cells from C57BL/6 mice, but its expression was remarkably induced upon T-cell activation. In the promoter of proximal Runx1, a highly conserved region was identified which spans from -412 to the transcription start site and harbors a NFAT binding site. In a luciferase reporter assay, this region was found to be responsive to T-cell activation through Lck and calcineurin pathways. Mutagenesis studies and chromatin immunoprecipitation assay indicated that the NFAT site was essential for NFAT binding and transactivation of the proximal Runx1 promoter. Furthermore, TCR signaling-induced expression of proximal Runx1 was blocked by treatment of cells with cyclosporin A. Together, these results demonstrate that the calcineurin-NFAT pathway regulates proximal Runx1 transcription upon TCR stimulation.

Identification of a novel fusion gene involving RUNX1 and the antisense strand of SV2B in a BCR‐ABL1‐positive acute leukemia

RUNX1, a key regulator of hematopoiesis, is frequently mutated or implicated in chromosomal translocations in acute leukemia. About half of RUNX1 translocations remain uncharacterized at the molecular level. We describe here one such event, a t(15;21)(q26.1;q22) translocation identified in an adult patient diagnosed with a t(9;22)(q34;q11.2)-positive acute leukemia. This previously unreported rearrangement yields a fusion of RUNX1 with the antisense strand of the SV2B gene, a new translocation partner of RUNX1, resulting in the expression of out-of-frame mRNA chimeric transcripts and the production of putative truncated RUNX1 isoforms. The t(15;21) translocation also dissociates the P1 promoter of RUNX1 from its open reading frame, reducing RUNX1 expression levels in the patient's leukemic cells. Our data suggest that RUNX1 haploinsufficiency collaborates with the BCR-ABL1 oncogene in this leukemia. The description of this atypical gene fusion is an important addition to the characterization of the pathogenomic mechanisms leading to RUNX1 structural and functional alterations. Furthermore, our data strongly suggests that inadequate dosage of this gene plays an essential role in leukemogenesis.

Expression of RUNX1 isoforms and its target gene BLK in childhood acute lymphoblastic leukemia

Bone marrow samples from children with acute lymphoblastic leukemia were analyzed for the expression of RUNX1a/b/c isoforms. Obtained patterns were associated with genetic abnormalities and the expression of the RUNX1 regulated gene BLK. RUNX1c was present in all patients, but the expected over-expression of RUNX1a was not observed. Over-expression of total RUNT domain isoforms was detected in patients with extra RUNX1 copies, and unexpectedly, in those with t(4;11). Only expression of the total RUNT domain-containing isoforms and BLK presented positive correlation. Results suggest a more complex role of RUNX1 in leukemogenesis than the proposed antagonism between the isoforms.

Tcf7 Is an Important Regulator of the Switch of Self-Renewal and Differentiation in a Multipotential Hematopoietic Cell Line

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34− cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34− cells, using RNA–Seq. We subsequently identified the target genes bound by TCF7, using ChIP–Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34− state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.

Neuroblastoma cell proliferation is sensitive to changes in levels of RUNX1 and RUNX3 protein

The Runt domain transcription factors Runx1 and Runx3 play crucial roles in dorsal root ganglion neurogenesis. Here we report that RUNX1 protein levels are maintained within a narrow range, despite highly variable levels of RUNX1 mRNA in neuroblastoma cell lines. Forced expression of the RUNX1 isoform AML1a (RUNX1/p27), a transcriptionally inactive competitive inhibitor, induces massive cell death, indicating that the function of RUNX1 is essential for neuroblastoma cell proliferation. Unexpectedly, over-expression of RUNX1 also induces cell death, as well as cell cycle arrest or differentiation. Furthermore, most neuroblastoma cell lines do not synthesize RUNX3 protein at detectable levels. Like RUNX1, exogenous expression also causes cell death, cell cycle arrest or differentiation. These results suggest that protein dosage of RUNX1 is critical for neuroblastoma cell growth and support a role for RUNX3 as a tumor suppressor in neuroblastoma.

CLCA2, a novel RUNX1 partner gene in a therapy-related leukemia with t(1;21)(p22;q22)

The RUNX1 gene is frequently rearranged in de novo and therapy-related leukemia. In the present study, we identified the CLCA2 gene as a novel fusion partner of RUNX1 in a case of therapy-related acute myeloid leukemia associated with t(1;21)(p22;q22). Reverse transcriptase–polymerase chain reaction analysis and sequencing revealed that the t(1;21) results in out-of-frame RUNX1-CLCA2 fusions. Alternative splicing generates at least six fusion transcripts, including a major transcript fusing RUNX1 exon 6 with CLCA2 exon 2. These out-of-frame fusions produce putative truncated RUNX1 isoforms retaining the DNA binding Runt domain but not the transcriptional regulatory domain of RUNX1. No mutations were found in the exons encoding the Runt and C-terminal domains of the nonrearranged RUNX1 gene. Similar to truncated RUNX1 isoforms previously described, these shortened products could act as dominant negative inhibitors of RUNX1-dependent transactivation. CLCA2 is a breast tumor suppressor gene that encodes a member of the calcium-activated chloride channel family and is involved for the first time in a chromosomal translocation. The RUNX1-CLCA2 fusion is another example of out-of-frame fusion generating truncated RUNX1 isoforms that represent a recurrent molecular mechanism in RUNX1-related leukemias.

Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells

RUNX1 (also known as acute myeloid leukemia 1) is an essential regulator of hematopoiesis and has multiple isoforms arising from differential splicing and utilization of two promoters. We hypothesized that the rare Runx1c isoform has a distinct role in hematopoietic stem cells (HSCs). We have characterized the expression pattern of Runx1c in mouse embryos and human embryonic stem cell (hESC)-derived embryoid bodies using in situ hybridization and expression levels in mouse and human HSCs by real-time polymerase chain reaction. We then determined the functional effects of Runx1c using enforced retroviral overexpression in mouse HSCs. We observed differential expression profiles of RUNX1 isoforms during hematopoietic differentiation of hESCs. The RUNX1a and RUNX1b isoforms were expressed consistently throughout hematopoietic differentiation, whereas the RUNX1c isoform was only expressed at the time of emergence of definitive HSCs. RUNX1c was also expressed in the AGM region of E10.5 to E11.5 mouse embryos, the region where definitive HSCs arise. These observations suggested that the RUNX1c isoform may be important for the specification or function of definitive HSCs. However, using retroviral overexpression to study the effect of RUNX1 isoforms on HSCs in a gain-of-function system, no discernable functional difference could be identified between RUNX1 isoforms in mouse HSCs. Overexpression of both RUNX1b and RUNX1c induced quiescence in mouse HSCs in vitro and in vivo. Although the divergent expression profiles of Runx1 isoforms during development suggest specific roles for these proteins at different stages of HSC maturation, we could not detect an important functional distinction in adult mouse HSCs using our assay systems.

The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis

AbstractThe transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatiotemporal expression is tightly regulated during embryonic development and is under the control of 2 alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.

Alternative Runx1 promoter usage in mouse developmental hematopoiesis

The interest in stem cell based therapies has emphasized the importance of understanding the cellular and molecular mechanisms by which stem cells are generated in ontogeny and maintained throughout adult life. Hematopoietic stem cells (HSCs) are first found in clusters of hematopoietic cells budding from the luminal wall of the major arteries in the developing mammalian embryo. The transcription factor Runx1 is critical for their generation and is specifically expressed at sites of HSC generation, prior to their formation. To understand better the transcriptional hierarchies that converge on Runx1 during HSC emergence, we have initiated studies into its transcriptional regulation. Here we systematically analyzed Runx1 P1 and P2 alternative promoter usage in hematopoietic sites and in sorted cell populations during mouse hematopoietic development. Our results indicate that Runx1 expression in primitive erythrocytes is largely P2-derived, whilst in definitive hematopoietic stem and/or progenitor cells from the yolk sac or AGM and vitelline and umbilical arteries both the distal P1 and proximal P2 promoters are active. After cells have migrated to the fetal liver, the P1 gradually becomes the main hematopoietic promoter and remains this into adulthood. In addition, we identified a novel P2-derived Runx1 isoform.

Specific Detection of CD56 (NCAM) Isoforms for the Identification of Aggressive Malignant Neoplasms with Progressive Development

Alternative splicing of transcripts from many cancer-associated genes is believed to play a major role in carcinogenesis as well as in tumor progression. Alternative splicing of one such gene, the neural cell adhesion molecule CD56 (NCAM), impacts the progression, inadequate therapeutic response, and reduced total survival of patients who suffer from numerous malignant neoplasms. Although previous investigations have determined that CD56 exists in three major isoforms (CD56(120kD), CD56(140kD), and CD56(180kD)) with individual structural and functional properties, neither the expression profiles nor the functional relevance of these isoforms in malignant tumors have been consistently investigated. Using new quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) strategies and novel CD56 isoform-specific antibodies, CD56(140kD) was shown to be exclusively expressed in a number of highly malignant CD56(+) neoplasms and was associated with the progression of CD56(+) precursor lesions of unclear malignant potential. Moreover, only CD56(140kD) induced antiapoptotic/proliferative pathways and specifically phosphorylated calcium-dependent kinases that are relevant for tumorigenesis. We conclude, therefore, that the specific detection of CD56 isoforms will help to elucidate their individual functions in the pathogenesis and progression of malignant neoplasms and may have a positive impact on the development of CD56-based immunotherapeutic strategies.

Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors

AbstractThe transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein–labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter–transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

Runx1 Deficiency Produces Aberrant Progenitor Self-Renewal through Selective Compromise of Pu.1 Mediated Transactivation but Not Transrepression.

A therapeutically crucial sub-set of leukemia cells are those that self-renew (leukemia stem-cells or LSC). LSC could be progenitors that abnormally self-renew from impairment of the terminal differentiation that usually regulates progenitor proliferation. In the present study, we demonstrate a molecular pathway that explains how Runx1 disruption (a frequent event in leukemia) can produce this phenotype, and its therapeutic implications. Hematopoietic differentiation is dependent on lineage-determining factors such as Pu.1. Pu.1 can repress or activate genes, depending on its interaction partners. One key interaction partner of Pu.1 is the non-lineage specific hematopoietic transcription factor Runx1, which is a frequent target of inherited and acquired mutations in myelodysplastic syndrome and leukemia. We demonstrate that knock-down of Runx1 in a hematopoietic model of inducible PU.1 function (PUERhi – gift of Harinder Singh), preserves the ability of Pu.1 to repress transcription of genes associated with self-renewal (Hoxb4, c-kit, Bmi-1). Instead, Runx1 deficiency specifically inhibited Pu.1 mediated upregulation of genes associated with differentiation (m-csfr, gm-csfr, F4/80). In chromatin-immunoprecipitation assays (ChIP), this failure in Pu.1 mediated gene transactivation was associated with decreased histone acetylation in the m-csfr and gm-csfr promoters which contain DNA binding motifs for Pu.1 and Runx1 in proximity. This suggests that Runx1 could regulate co-repressor/coactivator recruitment to Pu.1. Therefore, in co-immunoprecipitation and confocal assays, we examined the effects of RUNX1 on Pu.1 interactions with the co-repressors SIN3A and ETO2. RUNX1 prevented co-repressor recruitment to Pu.1. However, removal of the C-terminus transcription regulating region of RUNX1, or the replacement of this domain with ETO (modeling the leukemia fusion protein RUNX1-ETO), allowed formation of a RUNX1/Pu.1/co-repressor tri-partite complex. These findings, taken together with the histone acetylation changes noted at Pu.1/Runx1 composite DNA-binding sites upon Runx1 knock-down, suggest that RUNX1, but not RUNX1 isoforms that lack the C-terminal transcription regulating domain, prevent co-repressor recruitment to a promoter specific Pu.1/RUNX1 complex. This explains retained Pu.1 mediated transrepression, but impaired Pu.1 mediated transactivation, in the context of Runx1 deficiency or leukemia associated RUNX1 abnormalities. In murine models with Runx1 disruption, one feature is an increase in myeloid progenitors but decrease in stem-cells. Our findings show that Runx1 deficiency allows some phenotype transition from stem-cell to progenitor through intact Pu.1 repression of pro-self-renewal genes, but aberrant histone deacetylation prevents differentiation gene expression and terminal differentiation. Therefore, we examined if histone deacetylase inhibitor (HDACi) treatment could renew terminal differentiation of the Runx1 deficient cells. HDACi renewed terminal differentiation of the abnormally proliferating cells. In contrast, HDACi increased self-renewal of wild-type cells by preventing Pu.1 mediated repression of pro-self-renewal genes, the initial step in Pu.1 mediated terminal differentiation. Thus, HDACi had opposite effects on abnormally self-renewing progenitors versus normal self-renewing stem-cells. These finding could explain the accumulation of progenitors, and possibly LSC, with Runx1 disruption, and the potential for LSC-specific therapy that spares normal HSC.

Novel RUNX1 isoforms determine the fate of acute myeloid leukemia cells by controlling CD56 expression

AbstractCD56high acute myeloid leukemias (AMLs) have a poor prognosis, but it has been unclear how CD56 expression is controlled and how it relates to clinical aggressiveness. We show that CD56 expression on AML cells correlates with an abnormal expression pattern of runt-related transcription factor 1 (RUNX1) isoforms. Whereas full-length p48 RUNX1 (p48) up-regulated CD56 in AML cells, 3 previously unknown shorter RUNX1 isoforms, p38a, p30, and p24, suppressed CD56 expression. Both p48 and CD56 induced nuclear translocation of nuclear factor (NF)–κB and increased bcl2L12 expression, and inhibition of this pathway by small inhibitory RNA-mediated p48 knock down or NF-κB blockade substantially increased apoptosis in CD56+ AML cell lines. These findings indicate the potential for new therapy of CD56high AML by suppression of the “overactive” RUNX1/CD56/NF-κB signaling pathway(s).

Multifunctional reversible knockout/reporter system enabling fully functional reconstitution of the AML1/Runx1 locus and rescue of hematopoiesis

Mice deficient in the runt homology domain transcription factor Runx1 die of severe anemia in utero by embryonic day (E)12.5. A reactivatable Runx1 knockout embryonic stem cell (ESC) and mouse systems were generated by the targeted insertion of a loxP-flanked multipartite gene stop/trap cassette designed to simultaneously ablate the expression of Runx1 and report on the activity of its promoters. The cassette's in-frame LacZ reporter enabled activities of the proximal and the distal promoters to be differentially monitored. Although Runx1-null ESCs were capable of primitive erythroid differentiation in vitro, their capacity to generate granulocyte/macrophage or mixed myelo-erythroid embryoid bodies was lost. Cre-mediated reactivation restored Runx1 structural integrity and rescued the hematopoietic differentiation potential of ESCs. Mice with the reactivated allele survived, showed no hematopoietic deficit, and expressed all major splice isoforms of Runx1 appropriately. This multipurpose mouse model will be useful for the analysis of the critical Runx1-dependent check-point(s) in hematopoietic development.

Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.

Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.

Runx1/AML1/Cbfa2 Mediates Onset of Mesenchymal Cell Differentiation Toward Chondrogenesis

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.