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The AML1-ETO fusion protein is generated from the 8;21 chromosome translocation that is commonly identified in acute myeloid leukemia. AML1-ETO is a DNA binding transcription factor and has been demonstrated to play a critical role in promoting leukemogenesis. Therefore, it is important to define the molecular mechanism of AML1-ETO in the regulation of gene expression. Here, we report that the effect of AML1-ETO on the promoter of multidrug resistance-1 (MDR1) gene, a known AML1-ETO target, is highly cell type specific. Besides observing repression of the MDR1 promoter in C33A and CV-1 cells as reported previously, AML1-ETO strongly activated the promoter in K562 and B210 cells. More importantly, this activation required both the AML1 and ETO portions of the fusion protein, but did not depend on the AML1 binding site in MDR1 promoter. Furthermore, results from promoter deletion analysis and chromatin immunoprecipitation assays suggested that this activation effect was likely through the influence of the general transcription machinery rather than promoter-specific factors. Based on these data, we propose that AML1-ETO may have opposing effects on gene expression depending on the various conditions of the cellular environment.

The t(8;21)(q22;q22) translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in 15% of all cases of AML, including 40-50% of FAB M2 subtype and rare cases of M0, M1 and M4 subtypes. The most commonly known AML1-ETO fusion protein (full-length AML1-ETO) from this translocation has 752 amino acids and contains the N-terminal portion of RUNX1 (also known as AML1, CBFalpha2 or PEBP2alphaB), including its DNA binding domain, and almost the entire RUNX1T1 (also known as MTG8 or ETO) protein. Although alterations of gene expression and hematopoietic cell proliferation have been reported in the presence of AML1-ETO, its expression does not lead to the development of leukemia. Here, we report the identification of a previously unknown alternatively spliced isoform of the AML1-ETO transcript, AML1-ETO9a, that includes an extra exon, exon 9a, of the ETO gene. AML1-ETO9a encodes a C-terminally truncated AML1-ETO protein of 575 amino acids. Expression of AML1-ETO9a leads to rapid development of leukemia in a mouse retroviral transduction-transplantation model. More importantly, coexpression of AML1-ETO and AML1-ETO9a results in the substantially earlier onset of AML and blocks myeloid cell differentiation at a more immature stage. These results indicate that fusion proteins from alternatively spliced isoforms of a chromosomal translocation may work together to induce cancer development.

Normal blood-cell differentiation is controlled by regulated gene expression and signal transduction. Transcription deregulation due to chromosomal translocation is a common theme in hematopoietic neoplasms. AML1-ETO, which is a fusion protein generated by the 8;21 translocation that is commonly associated with the development of acute myeloid leukemia, fuses the AML1 runx family DNA-binding transcription factor to the ETO corepressor that associates with histone deacetylase complexes. Analyses have demonstrated that AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. Here, we report that the loss of the molecular events of AML1-ETO C-terminal NCoR/SMRT-interacting domain transforms AML1-ETO into a potent leukemogenic protein. Contrary to full-length AML1-ETO, the truncated form promotes in vitro growth and does not obstruct the cell-cycle machinery. These observations suggest a previously uncharacterized mechanism of tumorigenesis, in which secondary mutation(s) in molecular events disrupting the function of a domain of the oncogene promote the development of malignancy.

Although most solid tumors contain inactivating mutations of the p53 tumor suppressor, hematological malignancies do not contain frequent alterations in the p53 gene (

Background Sarilumab is the first fully-human monoclonal antibody directed against the IL-6R and is being investigated for the treatment of RA. Patients with RA have elevated tissue turnover at sites of inflammation, resulting from increased activity of proteases, including matrix metalloproteinase (MMP). MMP-cleaved fragments of collagen type 1 (C1M), collagen type 2 (C2M), collagen type 3 (C3M), and C-reactive protein (CRPM) are elevated in sera from patients with RA and reductions may be associated with treatment response. Indeed, the C1M neo-epitope has been recently identified as a potentially prognostic biomarker of bone erosion. Objectives The objective of this study was to determine whether sarilumab reduces the levels of serum biomarkers related to synovial inflammation and joint damage in patients with RA. Methods MOBILITY part A was the dose-ranging phase 2 portion of the MOBILITY trial of subcutaneous sarilumab in patients with active RA treated with concomitant methotrexate (MTX). Sera were collected at baseline, 2 weeks, and 12 weeks from patients randomized to treatment with MTX+placebo (Pbo), MTX+sarilumab 150 mg q2w, or MTX+sarilumab 200 mg q2w. C1M, C2M, C3M and CRPM were analyzed by ELISA. Results Baseline levels of each biomarker were similar among treatment groups. Sarilumab treatment at both doses resulted in marked reduction in levels of C1M, C2M, C3M and CRPM from baseline at 2 weeks (Table, percent change from baseline ± SE) and suppression of each marker was maintained at 12 weeks (data not shown). In contrast, in the MTX+Pbo group there was no significant decrease in these biomarkers at both 2 and 12 weeks. Observed AEs were similar to those reported with other IL-6 inhibitors. Conclusions Sarilumab treatment in patients with RA resulted in significant dose-dependent reduction in MMP-generated neo-epitope biomarkers related to joint and tissue turnover. The most dramatic reduction associated with sarilumab treatment was observed with the C1M neo-epitope, a potentially prognostic biomarker of structural damage (ref). Additional studies are needed to determine if reduction of MMP-cleaved fragments, such as C1M or CRPM, predict clinical and/or radiographic responses (bone erosion and/or joint space narrowing) in patients treated with sarilumab. References AS Siebuhr et al. Serological identification of fast progressors of structural damage with rheumatoid arthritis. Arthritis Research & Therapy 2013, 15:R86 doi:10.1186/ar4266. Acknowledgements Sponsored by Sanofi and Regeneron Pharmaceuticals Inc. (NCT01061736). Disclosure of Interest : S. Fiore Shareholder of: Sanofi US, Employee of: Sanofi US, A. Boyapati Shareholder of: Regeneron Pharmaceuticals, Inc., Employee of: Regeneron Pharmaceuticals, Inc., S. Hamon Shareholder of: Regeneron Pharmaceuticals, Inc., Employee of: Regeneron Pharmaceuticals, Inc., J. van Adelsberg Shareholder of: Regeneron Pharmaceuticals, Inc., Employee of: Regeneron Pharmaceuticals, Inc., M. Liu Shareholder of: Regeneron Pharmaceuticals, Inc., Employee of: Regeneron Pharmaceuticals, Inc., J. Hamilton Shareholder of: Regeneron Pharmaceuticals, Inc., Employee of: Regeneron Pharmaceuticals, Inc. DOI 10.1136/annrheumdis-2014-eular.3852

AML1-ETO is generated from t(8;21)(q22;q22), which is commonly associated with acute myeloid leukemia (AML), especially FAB M2 AML. While full length AML1-ETO fails to promote leukemia due to its detrimental effects on cell proliferation, an alternatively spliced isoform without its C-terminal NHR3 and NHR4 domains strongly induces leukemia. The NHR4 domain has a zinc-chelating topology and has been implicated in transcriptional repression through recruitment of the NCoR/SMRT complexes. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, suggesting that the NHR4 domain is responsible for generating inhibitory effects on leukemia development. To identify the molecular mechanism of this leukemogenic effect, we searched for NHR4-interacting factors with yeast two hybrid screening, and identified a novel NHR4-interacting protein, SON, a potential DNA/RNA-binding protein. The N-terminal fragment of SON with 81 amino acids, which is encoded by exons 1 and 2, was sufficient to interact with the NHR4 domain of ETO. Interaction of endogenous SON and AML1-ETO was also detected in t(8;21) AML cell lines. A serine substitution of cysteine 663 which disrupts the zinc-chelating topology of the NHR4 domain completely abolished its interaction with SON, while retaining the interaction with another ETO-binding protein, N-CoR. Knockdown of SON by siRNA resulted in significant growth arrest. Disruption of the interaction between AML1-ETO and endogenous SON by overexpressing the SON N-terminal fragment rescued cells from AML1-ETO-induced growth arrest. These results suggest that SON is an indispensable factor for cell growth and the interaction between endogenous SON and AML1-ETO generates negative effects on cell growth. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full length AML1-ETO. Taken together, these results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis and revealed the interaction of the NHR4 domain with SON, an essential protein for normal cell growth.

t(8;21) is a common chromosome translocation in acute myeloid leukemia (AML). AML1-ETO is the fusion protein produced by t(8;21). Results from our work and others indicate that full length AML1-ETO is not leukemogenic and needs additional mutations to promote leukemia. Most recently, we have identified an alternatively spliced form of AML1-ETO, AML1-ETO9a, that lacks the C-terminal NHR3 and NHR4 domains of AML1-ETO. Here, we report that unlike the negative effect of full length AML1-ETO in cell cycle progression, AML1-ETO9a enhances cell proliferation by shortening G1 phase of cell cycle in the IL-3 dependent multipotent hematopoietic progenitor cell line FDCPmix. Using retroviral mediated gene expression in murine hematopoietic cells and bone marrow transplantation in mice, we have demonstrated that AML1-ETO9a is highly leukemogenic. Most importantly, we show that AML1-ETO9a expression in transgenic MRP-8 AML1-ETO hematopoietic cells results in earlier onset of AML than AML1-ETO9a expression alone controls. Furthermore, the presence of full length AML1-ETO also alters the phenotype of leukemia progenitor cells from CD34- to CD34+. These results suggest that the alternatively spliced form AML1-ETO9a cooperates with full length AML1-ETO in leukemogenesis when coexpressed in t(8;21) patients.

The 8;21 translocation occurs in approximately 8% of patients with acute myeloid leukemia. This chromosomal lesion results in the fusion of the AML1 (RUNX1) gene on chromosome 21 to the ETO (MTG8) gene on chromosome 8 and generates the AML1-ETO fusion protein. We identified a truncated form of AML1-ETO which lacks approximately 200 amino acids at the C-terminus of full length AML1-ETO and strongly induces leukemia development (Yan et al, PNAS101(49): 17186–91, 2004). This protein is almost identical to a natural splice form of AML1-ETO designated AML1-ETO9a, which is expressed in t(8;21) patients. To study the leukemogenic properties of the truncated form of AML1-ETO (AEtr), we examined cell cycle profiles to compare it to full length AML1-ETO. In contrast to full length AML1-ETO which induced growth arrest of K562 cells, AEtr did not inhibit cell proliferation. Interestingly, AEtr expressing cells continued to proliferate in the presence of microtubule toxins that normally activate the spindle checkpoint and arrest cells in prometaphase of mitosis. Evaluation of critical components of the spindle checkpoint revealed a significant decrease in BubR1 protein in AEtr cells compared to vector control cells. BubR1 is a potent inhibitor of the anaphase promoting complex and reduction of BubR1 by either siRNA approaches or gene deletion in mice results in aneuploidy. Cytogenetic analyses of leukemic splenocytes from mice transplanted with AEtr cells identified multiple chromosomal abnormalities, including chromosome loss and gain as well as translocations. Observations of chromosomal alterations in t(8;21) patients have been well documented and include loss of either sex chromosome in 70% of patients, deletions of chromosome 9, and trisomy 8. These results indicate that inactivation of the spindle checkpoint through BubR1 reduction may be an important event in the development of t(8;21) leukemias.

Previous analyses demonstrated that full length AML1-ETO requires additional mutagenic events to promote leukemia. Here we describe a novel shorter form of the AML1-ETO fusion protein (AML1-ETOtr), which lacks C-terminal NHR3 and NHR4 [NCoR/SMRT interacting zinc-finger (ZnF)] domains. Unlike full length AML1-ETO, expression of AML1-ETOtr results in rapid development of acute myeloid leukemia (AML) in mice. Interestingly, naturally occurring alternatively spliced AML1-ETO variants exist in t(8;21) AML patient samples. AML1-ETOexon9a that includes one additional exon Exon 9a and AML1-ETOintron5 that uses a polyadenylation signal in Intron 5 are two such variants. AML1-ETOexon9a is structurally similar to AML1-ETOtr. AML1-ETOintron5 lacks the NHR2 heterodimerization domain as well as missing the NHR3 and NHR4 domains. To elucidate their putative role in leukemia development, we performed retroviral transduction and transplantation experiments with AML1-ETOexon9a, AML1-ETOintron5, and two zinc-finger domain (NHR4) mutants [AML1-ETOdZnF (deleted ZnF) and AML1-ETOmZnF (mutated ZnF)]. All of the mice transplanted with AML1-ETOexon9a (n = 8), AML1-ETOdZnF (n = 7), and AML1-ETOmZnF (n = 12) expressing cells rapidly developed AML, displaying a similar phenotype to AML1-ETOtr. None of AML1-ETOintron5 (n = 8) mice showed leukemia. These results suggest that the NHR2 domain of AML1-ETO is required whereas the NHR4 NCoR/SMRT interacting zinc finger domain is inhibitory for leukemia development. These observations suggest a novel mechanism of leukemogenesis for the AML1-ETO gene that can transcribe both tumor suppressive and/or oncogenic chimeric protein(s) by alternative RNA splicing.

The 8;21(q22;q22) translocation is found in 12% of all AML cases, including 40–50% of AML M2 subtype and a small portion of M0, M1, and M4 subtypes. The translocation brings together the DNA sequence encoding the N-terminus of the AML1 (RUNX1) with nearly all of ETO (MTG8) to generate the AML1-ETO fusion protein. The most commonly known AML1-ETO fusion protein (full length AML1-ETO) is encoded by AML1 exons 1 to 4 and by ETO exons 2 to 11 and has 752 a.a. However, a recently discovered ETO exon 9a can provide a protein translation stop codon after the last amino acid encoded by exon 8. The predicted AML1-ETO protein using exon 9a (AML1-ETO9a) has 575 a.a. and does not include the NHR3 and NHR4 domains at the C-terminus of full length AML1-ETO. To demonstrate the presence of the AML1-ETO9a transcript, we performed RT-PCR using RNA prepared from t(8;21) patient samples. Exon 9a transcript was detected in all patient samples when PCR generated a 595 bp fragment with ETO exon 6 and exon 9 primers and was detected in seven of the 14 samples when PCR generated a 1381 bp fragment with AML1 exon 4 and ETO exon 9a primers, indicating the presence of AML1-ETO9a mRNA in patient samples. The predicted molecular weight of AML1-ETO9a protein with 575 a.a. is 63.8 kDa and a 64 kDa AML1-ETO fusion protein has been reported in both Kasumi-1 cells and t(8;21) leukemic blasts. We compared the effects of AML1-ETO and AML1-ETO9a on proliferation and differentiation of hematopoietic cells using FDCPmix cell line, which is an IL-3 dependent multipotent hematopoietic stem cell line. Both forms of fusion proteins inhibited the myeloid lineage differentiation when cells were cultured in the presence of GM-CSF and G-CSF. However, these fusion proteins showed opposite effects on cell cycle progression. AML1-ETO suppressed cell cycle with prolonged G0/G1; AML1-ETO9a enhanced cell cycle with shortened G0/G1. Expression of these fusion proteins in primary bone marrow cells showed a similar result. Most importantly, only AML1-ETO9a efficiently induced acute myeloid leukemia in mice. Thus, the observation that a shorter form of AML1-ETO is a dominant factor in t(8;21) related leukemogenesis exhibits a new paradigm in the understanding of t(8;21) tumorigenesis.

The fusion protein AML1-ETO created by the 8;21 translocation involved in de novo acute myeloid leukemia requires secondary mutational events to promote leukemia. Here, we report that the loss of the molecular events associated with AML1-ETO C-terminus including an NCoR/SMRT interacting domain transforms AML1-ETO into a potent leukemogenic protein in mice. Furthermore, we present evidence of aberrant protein expression of cell cycle regulators in a hematopoietic cell line expressing AML1-ETO compared to the leukemogenic truncated form of AML1-ETO (AML1-ETOtr). Our studies show that AML1-ETO and AML1-ETOtr are biochemically isolated from the same cellular compartments, that they can oligomerize, and that they can both efficiently immunoprecipitate the transcription factor Gfi-1. However, contrary to AML1-ETO, AML1-ETOtr does not promote growth arrest. Western analyses show that cell cycle promoting factors cyclin D3 and cyclin A are decreased by AML1-ETO, while their RNA levels remain the same. In addition, an increase in the cdk-inhibitor p21WAF1 is observed in the presence of both proteins, while only AML1-ETO induces high expression of the cdk-inhibitor p27KIP1. These changes are associated with a deregulation of the SCF ubiquitin E3 ligase component Skp2 that is involved in controlling the levels of both cyclins and cdk-inhibitors. These observations suggest that AML1-ETO has tumor suppressor activity. Additional mutations to bypass this effect can change it into an oncogenic protein. Therefore, our results lead to a new model of AML1-ETO in leukemogenesis, i.e., the gain/loss of function of cell cycle regulators to promote cell cycle progression or disruption of molecular events associated with AML1-ETO C-terminal NCoR/SMRT interacting domain are required for AML1-ETO involved leukemogenesis. The disrupted molecular events may include 1) the loss of factor(s) physically interacting with the C-terminal domain of AML1-ETO, 2) the alteration by mutagenesis of signaling pathways downstream of AML1-ETO C-terminal domain, and 3) the dysfunction within AML1-ETO C-terminal domain by truncations or mutations.