MTG16 (myeloid translocation gene on chromosome 16) and its related proteins, MTG8 and MTGR1, define a small family of transcriptional corepressors. These corepressors share highly conserved domain structures yet have distinct biological functions and tissue specificity. In vivo studies have shown that, of the three MTG corepressors, MTG16 is uniquely important for the regulation of hematopoietic stem/progenitor cell (HSPC) proliferation and differentiation. Apart from this physiological function, MTG16 is also involved in carcinomas and leukemias, acting as the genetic target of loss of heterozygosity (LOH) aberrations in breast cancer and recurrent translocations in leukemia. The frequent involvement of MTG16 in these disease etiologies implies an important developmental role for this transcriptional corepressor. Furthermore, mounting evidence suggests that MTG16 indirectly alters the disease course of several leukemias via its regulatory interactions with a variety of pathologic fusion proteins. For example, a recent study has shown that MTG16 can repress not only wild-type E2A-mediated transcription, but also leukemia fusion protein E2A-Pbx1-mediated transcription, suggesting that MTG16 may serve as a potential therapeutic target in acute lymphoblastic leukemia expressing the E2A-Pbx1 fusion protein. Given that leukemia stem cells share similar regulatory pathways with normal HSPCs, studies to further understand how MTG16 regulates cell proliferation and differentiation could lead to novel therapeutic approaches for leukemia treatment.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype that is untreatable with hormonal or HER2-targeted therapies and is also typically unresponsive to checkpoint-blockade immunotherapy. Within the tumor microenvironment dysregulated immune cell metabolism has emerged as a key mechanism of tumor immune-evasion. We have discovered that the Liver-X-Receptors (LXRα and LXRβ), nuclear receptors known to regulate lipid metabolism and tumor-immune interaction, are highly activated in TNBC tumor associated myeloid cells. We therefore theorized that inhibiting LXR would induce immune-mediated TNBC-tumor clearance. Here we show that pharmacological inhibition of LXR activity induces tumor destruction primarily through stimulation of CD8+ T-cell cytotoxic activity and mitochondrial metabolism. Our results imply that LXR inverse agonists may be a promising new class of TNBC immunotherapies.

CBFA2T3 is a master transcriptional coregulator in hematopoiesis. In this study, we report novel functions of CBFA2T3 in acute myeloid leukemia (AML) relapse. CBFA2T3 regulates cell-fate genes to establish gene expression signatures associated with leukemia stem cell (LSC) transformation and relapse. Gene set enrichment analysis showed that CBFA2T3 expression marks LSC signatures in primary AML samples. Analysis of paired primary and relapsed samples showed that acquisition of LSC gene signatures involves cell type–specific activation of CBFA2T3 transcription via the NM_005187 promoter by GCN5. Short hairpin RNA–mediated downregulation of CBFA2T3 arrests G1/S cell cycle progression, diminishes LSC gene signatures, and attenuates in vitro and in vivo proliferation of AML cells. We also found that the RUNX1-RUNX1T1 fusion protein transcriptionally represses NM_005187 to confer t(8;21) AML patients a natural resistance to relapse, whereas lacking a similar repression mechanism renders non–core-binding factor AML patients highly susceptible to relapse. These studies show that 2 related primary AML-associated factors, the expression level of CBFA2T3 and the ability of leukemia cells to repress cell type–specific CBFA2T3 gene transcription, play important roles in patient prognosis, providing a paradigm that differential abilities to repress hematopoietic coregulator gene transcription are correlated with patient-specific outcomes in AML.

CBFA2/RUNX1 partner transcriptional co-repressor 3 (CBFA2T3, also known as MTG16 or ETO2) is a myeloid translocation gene family protein that functions as a master transcriptional corepressor in hematopoiesis. Recently, it has been shown that CBFA2T3 maintains leukemia stem cell gene expression and promotes relapse in acute myeloid leukemia (AML). However, a role for CBFA2T3 in myeloid differentiation of AML has not been reported. Here, we show that CBFA2T3 represses retinoic acid receptor (RAR) target gene expression and inhibits all-trans-retinoic acid (ATRA)-induced myeloid differentiation of AML cells. ChIP-Seq revealed that CBFA2T3 targets the RARα/RXRα cistrome in U937 AML cells, predominantly at myeloid-specific enhancers associated with terminal differentiation. CRISPR/Cas9-mediated abrogation of CBFA2T3 resulted in spontaneous and ATRA-induced activation of myeloid-specific genes in a manner correlated with myeloid differentiation. Importantly, these effects were reversed by CBFA2T3 re-expression. Mechanistic studies showed that CBFA2T3 inhibits RAR target gene transcription by acting at an early step to regulate histone acetyltransferase recruitment, histone acetylation, and chromatin accessibility at RARα target sites, independently of the downstream, RAR-mediated steps of transcription. Finally, we validated the inhibitory effect of CBFA2T3 on RAR in multiple AML subtypes and patient samples. To our knowledge, this is the first study to show that CBFA2T3 down-regulation is both necessary and sufficient for enhancing ATRA-induced myeloid gene expression and differentiation of AML cells. Our findings suggest that CBFA2T3 can serve as a potential target for enhancing AML responsiveness to ATRA differentiation therapies.

In up to 15% of acute myeloid leukemias (AMLs), a recurring chromosomal translocation, termed t(8;21), generates the AML1–eight–twenty-one (ETO) leukemia fusion protein, which contains the DNA-binding domain of Runt-related transcription factor 1 (RUNX1) and almost all of ETO. RUNX1 and the AML1–ETO fusion protein are coexpressed in t(8;21) AML cells and antagonize each other's gene-regulatory functions. AML1–ETO represses transcription of RUNX1 target genes by competitively displacing RUNX1 and recruiting corepressors such as histone deacetylase 3 (HDAC3). Recent studies have shown that AML1–ETO and RUNX1 co-occupy the binding sites of AML1–ETO–activated genes. How this joined binding allows RUNX1 to antagonize AML1–ETO–mediated transcriptional activation is unclear. Here we show that RUNX1 functions as a bona fide repressor of transcription activated by AML1–ETO. Mechanistically, we show that RUNX1 is a component of the HDAC3 corepressor complex and that HDAC3 preferentially binds to RUNX1 rather than to AML1–ETO in t(8;21) AML cells. Studying the regulation of interleukin-8 (IL8), a newly identified AML1–ETO–activated gene, we demonstrate that RUNX1 and HDAC3 collaboratively repress AML1–ETO–dependent transcription, a finding further supported by results of genome-wide analyses of AML1–ETO–activated genes. These and other results from the genome-wide studies also have important implications for the mechanistic understanding of gene-specific coactivator and corepressor functions across the AML1–ETO/RUNX1 cistrome.

BACKGROUND Nearly 15% of acute myeloid leukemia (AML) cases are caused by aberrant expression of AML1-ETO, a fusion protein generated by the t(8;21) chromosomal translocation. Since its discovery, AML1-ETO has served as a prototype to understand how leukemia fusion proteins deregulate transcription to promote leukemogenesis. Another leukemia fusion protein, E2A-Pbx1, generated by the t(1;19) translocation, is involved in acute lymphoblastic leukemias (ALLs). While AML1-ETO and E2A-Pbx1 are structurally unrelated fusion proteins, we have recently shown that a common axis, the ETO/E-protein interaction, is involved in the regulation of both fusion proteins, underscoring the importance of studying protein–protein interactions in elucidating the mechanisms of leukemia fusion proteins. OBJECTIVE In this review, we aim to summarize these new developments while also providing a historic overview of the related early studies. METHODS A total of 218 publications were reviewed in this article, a majority of which were published after 2004.We also downloaded 3D structures of AML1-ETO domains from Protein Data Bank and provided a systematic summary of their structures. RESULTS By reviewing the literature, we summarized early and recent findings on AML1-ETO, including its protein–protein interactions, transcriptional and leukemogenic mechanisms, as well as the recently reported involvement of ETO family corepressors in regulating the function of E2A-Pbx1. CONCLUSION While the recent development in genomic and structural studies has clearly demonstrated that the fusion proteins function by directly regulating transcription, a further understanding of the underlying mechanisms, including crosstalk with other transcription factors and cofactors, and the protein–protein interactions in the context of native proteins, may be necessary for the development of highly targeted drugs for leukemia therapy.

No abstract

Background:Roughly 15% of AML cases are derived from the t(8;21) translocation, a molecular event that results in the production of the AML1-ETO (AE) fusion transcription factor. This aberrant protein induces a broad dysregulation of the transcriptome and causes expansion of leukemia stem cells (LSCs), which predisposes clones to an increased risk of second-hit mutations. In isolation, without the presence of type I mutations, the t(8;21) mark is considered to be one of favorable prognosis. However, a number of LSC characteristics have been shown to influence patient survival, including expression levels of the cell surface glycoprotein, CD34, and of the truncated isoform of AE, AML1-ETO9a (AE9a). Despite these findings, a unified model of how these factors interact to further the progression of t(8;21) AML has not been determined. Here, we use a combination of in vitro assays and bioinformatic analyses of patient datasets with the hopes of identifying an underlying connection between independent, prognostic factors. Results: We first performed gene knockdown studies with shRNA constructs (against AE) in a pKLO.1 backbone, which was transduced into the AML-model cell line Kasumi-1. Subsequent RT-PCR showed a large decrease in CD34 expression and, serving as a control, an increase in RASSF2, a cytoplasmic signaling protein normally repressed by AE. This led us to perform gene overexpression of cDNA encoding AE or AE9a into CD34-enriched cord blood HSCs. Interestingly, we were able to show that both AE and AE9a increased the expression of CD34, and AE9a was able to do so to a greater extent. This prompted us to analyze previous ChIP-seq data from Kasumi-1 cells, and we discovered high levels of AE binding to an upstream enhancer of CD34. While this site was free of any E-box binding motifs, we nonetheless observed binding peaks of the E-protein HEB, and further gene knock-down studies showed that HEB recruitment to this site depended on AE and, in turn, CD34 expression depended on HEB recruitment. Next, we used differential gene expression analyses to determine the CD34-coexpressed genes from a publically available patient microarray dataset (GSE14468, n=516). CD34-high patient samples showed an accompanying enrichment of JUP, KIT, CD133, HEB, E2A and ETO2, and a decrease in immune signaling molecules such as IFNGR1 and IFNGR2. ETO2 is the only ETO family member significantly expressed in HSCs and is an important corepressor of E-proteins HEB and E2A; it is thought to help repress the pro-differentiation and anti-proliferation effects of E-proteins. ETO2, being an ETO family member, is also able to oligomerize with other ETO family members, such as ETO, and thus may play a role in modifying the functions of the AE-transcriptional complex. Intriguingly, we have evidence that AE and AE9a repress ETO2, although AE9a does so to a lessened extent. We then performed Kaplan-Meier analyses on subgroups of non-CBF AML and t(8;21) AML cases. Unsurprisingly, we found that both groups experienced lower event-free survival when CD34 and ETO2 were high (p=.028 and p=.040), and the t(8;21) group when IFNGR was low (p=.014). Additionally, although CD34 expression was higher in t(8;21) patients compared to non-CBF AML (p=.0027), ETO2 expression was lower (p=.025). If both subgroups were further divided into CD34-high and CD34-low groups, ETO2 expression was, in both cases, higher in the CD34-high patients and lower in the CD34-low patients, suggesting a possible regulatory link between these two genes. Finally, we performed ETO2 gene knockdown experiments in CD34+ HSCs and observed an increase in IFNGR1 and IFNGR2 expression. Conclusions: Our findings suggest a clinically relevant association between CD34, ETO2, and IFNGR in both t(8;21) and non-CBF AML. We believe this regulatory axis is especially relevant in relapse likelihood and may be hijacked by LSCs to preserve their "stemness" and resistance to therapy. Our findings may suggest that AE simultaneously activates CD34 and represses ETO2, providing explanation for why full-length AE is slow to induce leukemia in mouse models and a relatively good prognostic mark in humans. Additionally, our findings suggest a loss of ETO2repression as a potential mechanism by which the AE9a isoform, and perhaps other second-hit mutations, converts LSCs to a more dangerous phenotype. Supported by NIH grants R01HL093195-01A1 to JZ and T32 training grant T32GM008306-26A1 Disclosures No relevant conflicts of interest to declare.