AML1-ETO Fusion Protein Research Articles

CRISPR-Cas9 Screening Identifies Therapeutic Targets for AML1-ETO Positive Leukemia

Objectives: Targeted therapy has achieved tremendous success in leukemia therapy over the past few decades, especially in those with fusion proteins, such as t(15;17) which generates PML-RARa and t(9;22) which generates BCR-ABL. However, effective targeted therapy for AML1-ETO-positive leukemia, which arises from one of the most frequent chromosomal translocations t(8;21), remains largely elusive. In this study, we performed CRISPR-Cas9 screening on the AML1-ETO-positive Kasumi-1 cell line using an AML-related sgRNA library to identify potential therapeutic targets that could restore normal differentiation in AML1-ETO-positive AML. Methods: To identify critical genes capable of reversing differentiation blockage in AML1-ETO-driven leukemogenesis, we constructed an AML1-related sgRNA library which focused genes and signaling pathways regulated by AML1-ETO. This sgRNA library consist of (1) coding genes of proteins interacting with AML1, (2) genes activated by AML1-ETO, and (3) genes downregulated upon knockdown of AML1-ETO as observed in CHIP-seq results. This sgRNA library comprises 4130 sgRNAs targeting 929 human genes (~4 sgRNA/gene), along with 100 control sgRNAs. CD13 high and CD13 low populations were collected by flow cytometry at 10th day post-transduction of sgRNA library. The enrichment of gRNAs between CD13 high and CD13 low group was analyzed using the MAGeCK program, and the candidate genes were identified based on the log2 fold change of sgRNA abundance. To make the screening results more reliable, we used CD11b as another screening marker to repeat aforementioned process. We further preformed single candidate gene knockout in Kasumi-1 and SKNO-1 cell line to validate the screening phenotype. The regulatory function of candidate gene in AML1-ETO positive leukemia was explored through various in vitro assays, including cell apoptosis, cell proliferation, morphological evaluation, flow cytometry analysis, quantitative real-time PCR (qPCR), and Western blot. Results: The gene set enriched in the CD13 high group is considered to be positive hit. Silence of these genes may reverse blockage of differentiation, making them potential therapeutic targets for AML1-ETO leukemia. Additionally, genes such as CBFB, MTA2, DOT1L, PRMT1, RUNX1 and KDM1A, which had been previously reported to promote myeloid differentiation when down-regulated, were also significantly enriched in the CD13 high group, these findings demonstrated the credibility of our screening strategy and results. Clathrin Heavy Chain (CLTC) emerged as one of the top-ranking genes in our study. CLTC is a crucial protein involved in the regulation of clathrin-mediated endocytosis, which plays a critical role in various cellular processes including cell signaling, proliferation, and differentiation. Expression of CD13 and CD16 were upregulated in CLTC knockout cells, further validating our screening results. Besides, morphological analysis displayed a more mature cell morphology with a decrease in nucleus-to-cytoplasm ratio and indented shaped nuclei, suggesting that repression of CLTC promotes terminal differentiation of AML blasts. Cell biology experiments indicated that knockout of CLTC remarkably inhibited the proliferation of Kasumi-1 and SKNO-1 cells, meanwhile, apoptosis of these cells was accelerated. Therefore, these results collectively suggested that the down-regulation of CLTC exhibited significant tumor-suppressive effect in AML1-ETO leukemia. Furthermore, Western blot and RT-qPCR analyses revealed that CLTC is highly expressed in AML1-ETO-positive leukemia, whereas its expression is downregulated upon deletion of AML1-ETO fusion protein. This suggested that CLTC might be regulated by AML1-ETO fusion protein and contribute to its oncogenic activity. Conclusions: Our results revealed a possible novel mechanism underlying the blockage of myeloid differentiation in AML1-ETO leukemia, and have identified CLTC as a promising therapeutic target for AML1-ETO-driven leukemogenesis.

Pre-Leukemic Transformation of the Bone Marrow Microenvironment Induced By AML1-ETO Fusion Protein

Objective: The bone marrow (BM) microenvironment, especially stromal cells (SCs) and cytokines, plays a critical role in supporting normal hematopoiesis. However, in cases of hematological malignancies such as acute myeloid leukemia (AML), leukemia cells can reshape the BM microenvironment to promote their survival. Despite this, there is still limited understanding of the changes in AML1-ETO fusion protein induced pre-leukemia microenvironment niche. In this study, the alterations of the BM microenvironment in the Aml1 Eto/+; Mx1-Cre (AE KI) mouse model are thoroughly explored and analyzed. This investigation will encompass the study of SCs, cytokines, their sources, and their potential impacts on the occurrence and progression of AML induced by AML1-ETO. Methods: After 4 weeks of PIPC induction, proportion changes of endothelial cells (ECs), osteoblasts, mesenchymal stem cells (MSCs) in the BM of Aml1 Eto/+; Mx1-Cre mice (experimental group) and Aml1 Eto/+; w/o Mx1-Cre mice (control group) were analyzed by flow cytometry. Bulk RNA sequencing (RNAseq) and single cell RNA sequencing (scRNAseq) techniques were applied for analysis of abnormal hematopoietic Lin -Sca-1 +c-Kit + (LSK) stem cells. Cytokine levels were detected using ELISA and Luminex bead assay. Additionally, CRISPR-Cas9 technology was used to knock out the target genes and their functions were validated. Results: Following 4 weeks of PIPC induction, the experimental group exhibited a considerable reduction in the proportion and absolute number of ECs, osteoblasts and MSCs, with ECs being the most significantly reduced. The Luminex assay revealed a substantial increase in cytokine release in the BM of the experimental group, including IL4, IL10, TNFa, Cxcl10, Cxcl11, Ccl1, Ccl2, Ccl4, Ccl7, etc. Bulk RNAseq studies of LSK cells showed that the inflammatory pathway in the experimental group was significantly upregulated. To further explore the release sources of cytokines in the BM and heterogeneity in LSK cell cytokine secretion, scRNAseq analysis of LSK cells was conducted in the AE KI group, which were classified into 9 subgroups. Subgroups 1 and 5 exhibited a significant upregulation of Ccl2, Ccl4, Cxcl2, and IL15. Subgroups 4 and 8 showed upregulation of Ccl2 and IL4. Subgroups 0 and 2 exhibited upregulation of Ccl4, while subgroups 6, 7, and 9 showed upregulation of Cxcl2. Notably, subgroups 1 and 5 also displayed increased expression of Ccr5, which is the receptor for Ccl2. To further validate the origins of cytokines, LSK cells were sorted from experimental group and the control group for in vitro culture, respectively. Subsequently, the cell culture supernatants were collected for Luminex assay, revealing a notable upregulation of Ccl2 and Ccl7 in the experimental group. Furthermore, when comparing m-cherry + LSK cells (AML1-ETO positive) with m-cherry - LSK cells (AML1-ETO negative) from the same mouse, a significant increase in the Ccl2 levels in the m-cherry + LSK cell culture supernatant was observed. Flow cytometry analysis of LSK cells further revealed high expression of Ccl2 and its receptor Ccr5 in Ccl2 + LSK cells. In order to gain a deeper understanding of the functions of Ccl2 and Ccl7, CRISPR-Cas9 technology was used to knockout Ccl2 and Ccl7 in c-kit + cells, which were then transplanted into mice for further investigation, respectively. After 3 months, partial restoration of ECs was observed compared to that of the scramble control group. Conclusion: Our findings suggested that significant changes occurred in the BM microenvironment in AML1-ETO-induced pre-leukemia stage. These changes were characterized by a decrease in SCs and an increase in pro-inflammatory cytokine levels. Moreover, our study identified the heterogeneity in cytokine transcriptional profiles among AE KI LSK cells. The secretion of Ccl2 and Ccl7 by AE KI LSK cells with a blockage in hematopoietic differentiation seemed to be involved in the reduction of stromal cell population. These results provided valuable insights for further exploration of the intricate interactions between abnormal hematopoietic stem cells and the BM microenvironment in AML1-ETO-induced pre-leukemic and leukemic states. Understanding these interactions is crucial for advancing our knowledge of the underlying mechanisms involved in the development and progression of AML.

ABL1 kinase as a tumor suppressor in AML1-ETO and NUP98-PMX1 leukemias

Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98 fusion proteins. Abl1−/− murine hematopoietic cells transduced with AML1-ETO and NUP98-PMX1 gained proliferation advantage when compared to Abl1 + /+ counterparts. Conversely, overexpression and pharmacological stimulation of ABL1 kinase resulted in reduced proliferation. To pinpoint mechanisms facilitating the transformation of ABL1-deficient cells, Abl1 was knocked down in 32Dcl3-Abl1ko cells by CRISPR/Cas9 followed by the challenge of growth factor withdrawal. 32Dcl3-Abl1ko cells but not 32Dcl3-Abl1wt cells generated growth factor-independent clones. RNA-seq implicated PI3K signaling as one of the dominant mechanisms contributing to growth factor independence in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib exerted selective activity against Lin-cKit+ NUP98-PMX1;Abl1−/− cells when compared to the Abl1 + /+ counterparts. Since the role of ABL1 in DNA damage response (DDR) is well established, we also tested the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi). AML1-ETO;Abl1−/− and NUP98-PMX1;Abl1−/− cells were hypersensitive to DNA-PKi and ATRi, respectively, when compared to Abl1 + /+ counterparts. Moreover, ABL1 kinase inhibitor enhanced the sensitivity to PI3K, DNA-PKcs and ATR inhibitors. In conclusion, we showed that ABL1 kinase plays a tumor suppressor role in hematological malignancies induced by AML1-ETO and NUP98-PMX1 and modulates the response to PI3K and/or DDR inhibitors.

LYL1 facilitates AETFC assembly and gene activation by recruiting CARM1 in t(8;21) AML

Transcription factors (TFs) play critical roles in hematopoiesis, and their aberrant expression can lead to various types of leukemia. The t(8;21) leukemogenic fusion protein AML1-ETO (AE) is the most common fusion protein in acute myeloid leukemia and can enhance hematopoietic stem cell renewal while blocking differentiation. A key question in understanding AE-mediated leukemia is what determines the choice of AE to activate self-renewal genes or repress differentiation genes. Toward the resolution of this problem, we earlier showed that AE resides in the stable AETFC complex and that its components colocalize on up- or down-regulated target genes and are essential for leukemogenesis. In the current study, using biochemical and genomic approaches, we show that AE-containing complexes are heterogeneous, and that assembly of the larger AETFC (containing AE, CBFβ, HEB, E2A, LYL1, LMO2, and LDB1) requires LYL1. Furthermore, we provide strong evidence that the LYL1-containing AETFC preferentially binds to active enhancers and promotes AE-dependent gene activation. Moreover, we show that coactivator CARM1 interacts with AETFC and facilitates gene activation by AETFC. Collectively, this study describes a role of oncoprotein LYL1 in AETFC assembly and gene activation by recruiting CARM1 to chromatin for AML cell survival.

A direct comparison between AML1-ETO and ETO2-GLIS2 leukemia fusion proteins reveals context-dependent binding and regulation of target genes and opposite functions in cell differentiation.

The ETO-family transcriptional corepressors, including ETO, ETO2, and MTGR1, are all involved in leukemia-causing chromosomal translocations. In every case, an ETO-family corepressor acquires a DNA-binding domain (DBD) to form a typical transcription factor—the DBD binds to DNA, while the ETO moiety manifests transcriptional activity. A directly comparative study of these “homologous” fusion transcription factors may clarify their similarities and differences in regulating transcription and leukemogenesis. Here, we performed a side-by-side comparison between AML1-ETO and ETO2-GLIS2, the most common fusion proteins in M2-and M7-subtypes of acute myeloid leukemia, respectively, by inducible expression of them in U937 leukemia cells. We found that, although AML1-ETO and ETO2-GLIS2 can use their own DBDs to bind DNA, they share a large proportion of genome-wide binding regions dependent on other cooperative transcription factors, including the ETS-, bZIP- and bHLH-family proteins. AML1-ETO acts as either transcriptional repressor or activator, whereas ETO2-GLIS2 mainly acts as activator. The repressor-versus-activator functions of AML1-ETO might be determined by the abundance of cooperative transcription factors/cofactors on the target genes. Importantly, AML1-ETO and ETO2-GLIS2 differentially regulate key transcription factors in myeloid differentiation including PU.1 and C/EBPβ. Consequently, AML1-ETO inhibits, but ETO2-GLIS2 facilitates, myeloid differentiation of U937 cells. This function of ETO2-GLIS2 is reminiscent of a similar effect of MLL-AF9 as previously reported. Taken together, this directly comparative study between AML1-ETO and ETO2-GLIS2 in the same cellular context provides insights into context-dependent transcription regulatory mechanisms that may underlie how these seemingly “homologous” fusion transcription factors exert distinct functions to drive different subtypes of leukemia.

Abstract 823: ABL1 kinase regulates leukemia phenotype and response to therapy

Abstract The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase. Cytoplasmic ABL1 stimulates, whereas nuclear ABL1 inhibits cell growth. Analysis of the CGAP Mitelman database detected loss of ABL1 allele in hematologic malignancies. Among the top genetic aberrations detected in acute myeloid leukemias were t(8;21) generating fusion protein AML1-ETO and NUP98 translocations, e.g., t(1;11) encoding NUP98-PMX1.Expression of AML1-ETO and NUP98-PMX1 in Lin-cKit+ stem cell enriched bone marrow cells from Abl1-/-;Vav-Cre and Abl1+/+;Vav-Cre mice revealed that oncogene-expressing Abl1-/- cells gained tremendous proliferation advantage. Conversely, overexpression of ABL1 and DPH (ABL1 kinase agonist)-mediated activation of ABL1 kinase resulted in reduced proliferation of AML1-ETO and NUP98-PMX1 cells. Since the role of ABL1 in DNA damage response (DDR) is well established, we applied the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi) to identify potential therapeutic vulnerabilities. AML1-ETO;Abl1-/- cells were more sensitive to all three inhibitors when compared to AML1-ETO;Abl1+/+ cells. Remarkably, NUP98-PMX1;Abl1-/- cells were exceptionally sensitive to ATRi, but not to ATMi and DNA-PKi when compared to NUP98-PMX1;Abl+/+ cells. ABL1 did not affect the sensitivity of transformed cells to DNA damaging agent doxorubicin. To pinpoint mechanisms collaborating with Abl1 deletion in transformation of hematopoietic cells, Abl1 was knockout in 32Dcl3 cells by CRISPR/Cas9 to obtain 32Dcl3-Abl1ko cells. 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells were challenged to induce transformation by starving them from growth factors (IL3). Only 32Dcl3-Abl1ko cell populations generated growth factor-independent cells. This indicates that the absence of Abl1 may facilitate malignant transformation of hematopoietic cells. RNA-seq was performed to compare gene expression in 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells in the presence or absence of IL-3. Pathway analysis of FPKM reads (top 30 significant canonical pathways) and Venn diagram analysis followed by KEGG pathway analysis (291 genes uniquely expressed in growth factor-independent 32Dcl3-Abl1ko cells) implicated PI3K-AKT pathway signaling including mTOR and sirtuins (SITR) in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib, mTOR inhibitor rapamycin and SIRT2 inhibitor thiomyristoyl exerted selective activity against Lin-cKit+ AML1-ETO;Abl1-/- and/or NUP98-PMX1;Abl1-/- cells when compared to Abl1+/+ counterparts. Moreover, inhibition of ABL1 kinase by imatinib in Lin-cKit+ AML1-ETO;Abl1+/+ and/or NUP98-PMX1;Abl+/+ cells induced the sensitivity to PI3K-ATK pathway inhibitors. In conclusion, we showed that normal ABL1 kinase plays a tumor suppressor role and is a potential therapeutic target in hematological malignancies induced by AML1-ETO and NUP98-PMX1. The latter statement is being currently tested in animal models. Citation Format: Konstantin Golovine, Zhaorui Lian, Kumaraswamy Naidu Chitrala, Gleb Ablakov, Margaret Nieborowska-Skorska, Jian Huang, Tomasz Skorski. ABL1 kinase regulates leukemia phenotype and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 823.

Implantation of Patient-Derived Acute Leukemia Cells into the Hematopoietic Niche of Immunocompromised Mice Favors Faithful Recapitulation of the Disseminated Growth of Human Leukemia

The aim of this study was to determine the influence of the engraftment site on the tumor biology and drug sensitivity of a panel of hematological patient derived xenografts (PDX). PDX cells (3x10e6 cells/mouse) were injected intratibially (i.t.), intrasplenically (i.s.) or subcutaneously (s.c.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in bone marrow (BM), peripheral blood (PB) and spleen during the course and at the end of the experiment. Overall survival (OS) served as an additional read-out. In nine models, sensitivity towards cytarabine (Cy) was evaluated across the different implantation sites. Up to now our group has established 26 PDX of acute leukemia (23 AML, one ALL, two APL) covering a broad range of the different genetic subtypes: amongst other molecular alterations models carrying PML-RARA or AML1-ETO fusion proteins are included in the collection. 24/26 lines engrafted when injected i.t., 17/22 developed tumors after i.s. implantation and 16/19 established tumors post s.c. injection of T cell depleted PB or BM of patients. Hence, for the majority of the models the engraftment capacity per se was not dependent on the injection site. Yet, some models could exclusively be propagated in a specific setting: one AML model, grew solely i.t. or i.s., whereas another AML and one APL could be propagated only when injected s.c.. The implantation site did influence tumor growth behavior: Mean OS ranged from 161.3 (±22.3) days for i.t. to 93.1 (±13.1) days for s.c. propagation. I.s. transplanted mice had to be sacrificed after 137.6 (±25.2) days. The differences in mean OS between the three settings were not statically significant (one way ANOVA). Nevertheless, the OS times within single models differed significantly depending on the implantation technique (p ranging from < 0.005 - 0.017, Log-rank (Mantel-Cox) test). The dissemination pattern of individual lines was as well affected by the injection site. In general, infiltration of the investigated hematopoietic organs was significantly higher when cells were engrafted i.t. or i.s as compared to the s.c. approach (p< 0.0001, one way ANOVA). The mean BM (i.t.: 58.1 ± 8.1%; i.s.: 51.3 ± 13.4%; s.c.: < 0.01%), spleen (i.t.: 40.4 ± 8.8%; i.s.: 52 ± 11.5%; s.c.: < 0.01%) and PB (i.t.: 36.6 ± 8.1%; i.s.: 36.4 ± 12.0 %; s.c.: 10.4 ± 7.2 %) infiltration rate across all 26 models was similar in the i.t. and the i.s. setting but considerably higher than in the s.c approach. Significant differences could be detected between individual models depicting characteristic engraftment patterns independent of the injection site (p ranging from < 0.003 - 0.02, one way ANOVA). The expression pattern of the six investigated surface markers (CD45, CD3, CD34, CD33, CD38 & HLA-ABC) was not influenced by the application route. Every model depicted its distinct expression pattern similar to the expression pattern of the donor sample. Of note, the expression pattern was stable across different passages in the murine host. Cy was highly active in five AML and one ALL model: OS was significantly prolonged in the s.c. as well as in the disseminated setting (p ranging from < 0.001 - 0.003, Log-rank (Mantel-Cox) test). Three other AML models depicted a less pronounced sensitivity towards Cy (p ranging from < 0.005 - 0.007, Log-rank (Mantel-Cox) test) both growing s.c. or i.t.. Thus, drug sensitivity was not influenced by the injection site of the leukemic cells. To the best of our knowledge, this is the first time that such a thorough side-by-side comparison was performed in acute leukemia PDX models. Taken together, the application route has a major impact on the tumor biology of the PDX model: i.t. and i.s but not s.c. implantation induced a disseminated growth pattern mimicking consistently the human disease. Up to now, implanting leukemic cells i.t. led to the highest engraftment rate (92%) within the panel of established AML PDX models, indicating the importance of the hematopoietic niche as a supportive tumor microenvironment. Therefore, the establishment of favorable genetic AML subtypes should be preferably done by i.t. implantation. Nevertheless, the consistent expression of surface markers as well as sensitivity towards Cy independent of the implantation site, justifies the use of s.c. implanted AML PDX for screening approaches in an early stage of the drug development process. DisclosuresSchueler:Charles River Research Services Germany GmbH: Employment. Lenhard:Charles River Research Services Germany GmbH: Employment. Klingner:Charles River Research Services Germany GmbH: Employment. Oswald:Charles River Research Services Germany GmbH: Employment.

This Is a Title in Chidamide, a Novel HDAC Inhibitor, Is Effective for Treating Leukemia Relapse Post Allogeneic Stem Cell Transplantation and Results in Th1 Polarization in AML Patients with AML1-ETO

Background: Although acute myeloid leukemia (AML) with t(8; 21) carries a favorable prognosis, relapse still occursin up to 40% of patients who require allogeneic stem cell transplantation (alloSCT). However, 10-20% of this patients suffer post-transplant relapse. AML1-ETO fusion protein can cause leukemogenesis via epigenetic modulation of AML1 target genes. In addition, emerging evidence support a role of this fusion protein in the downregulation of immune modulators on leukemia and subsequent immune evasion. Both process are histone deacetylase (HDAC)-dependent. Therefore we hypothesize that inhibition of HDAC may represent an effective treatment for AML with AML1-ETO. Chidamide is a novel HDAC inhibitor that has been CFDA approved for PTCL. In this study, we aim to test the safety and efficacy of Chidamide in post-transplant relapse AML with AML1-ETO.Method: This is a single arm, open label phase I study administrating Chidamide in AML patients with AML1-ETO who has molecular relapse post alloSCT. Chidamide is given 0.5mg/kg PO twice a week for children or 30mg PO twice a week for adults. All patients are treated for 3 months before analysis of molecular study. Treatment is continued for patients with persistently positive AML1-ETO, while discontinued in patients who achieved complete molecular remission (CMR). For patients not able to achieve CMR, DLI is added in addition to Chidamide. The primary objective is to determine the safety of Chidamide in AML post-transplant. The secondary objective is to evaluate overall response. Blood and bone marrow samples were collected prior to the treatment, monthly during the treatment, and 3 months after discontinuation of the treatment. Correlative studies for immune markers were performed by flow cytometry.Results: Seven patients have been enrolled in this study. No severe adverse event was observed. The most common adverse event was CTCAE grade 2 thrombocytopenia (7/7). Other adverse events that were possibly or probably related to Chidamide included grade 1 constitutional symptoms (7/7), reversible grade 2 transaminitis (3/7), grade 1 renal dysfunction (1/7), grade 1 digestive tract symptoms (4/7), grade 2 neutropenia (2/7), and grade 1 skin rash (4/7). No GVHD was observed in patients received Chidamide alone, whereas 2 patients received Chidamide and DLI developed grade 3 GVHD. Strikingly all 7 patients responded to Chidamide with decreased AML1-ETO level (ORR 100%). Among them, 6 patients achieved complete response (CR 85.7%), 1 patient had partial response (PR 14.3%) and eventually deteriorated.We also performed correlative studies and made several important findings. First, we assessed the distribution of each immune component from these 7 patients prior to Chidamide treatment, at which point all patients had molecular relapse. Samples from 4 patients who remain CMR post-transplant were used as controls. We observed a significantly decreased absolute counts of CD3+CD4-CD8+T cells in the patients with relapse, compared with that of patients remaining remission (272.0±63.7 vs. 2087.5±536.5, P=0.042), whereas NK cells, B cells, and CD3+CD4+CD8- T cells showed no statistical difference. Importantly there was a significant increase in percentages of Th2 and Th17 cells, while no difference of Th1 and FOXP3+Treg cells was observed. We next examined the level of Th1, Th2, and Th17 from the 7 relapse patients prior to treatment, compared with that from the same patient 1, 2, or 3 months post Chidamide treatment. We found that both percentages and absolute counts of Th1 cells increased gradually post treatment (26.0±12.8 vs. 190.8±43.4, P=0.027). In contrast, Th2 and Th17 cells decreased. Interestingly, at the time of 3 months after Chidamide discontinuation, the increased absolute counts of Th1 cells and decreased percentages of Th2 and Th17 remained. Furthermore, the Th1/Th2 ratio was low in patients with relapse disease. It increased gradually during Chidamide treatment, correlated with the down trend of AML1-ETO titer.Conclusion: Chidamide is safe and tolerable in AML patients with AML1-ETO who has post-transplant relapse. Although limited by sample size, our study showed promising clinical efficacy of Chidamide in AML. Importantly our study demonstrated a polarization for Th1 and the significant elevated ratio of Th1/Th2 in patients received Chidamide, which may contribute to the treatment efficacy of Chidamide for AML patients with AML1-ETO. DisclosuresWang:Meryx: Equity Ownership; University of North Carolina: Patents & Royalties.

Characterization of the Post-Transcriptional Regulation of AML1-ETO Expression in t(8;21) Leukemia Cells

The translocation between chromosomes 8q22 and 21q22 [t(8;21)(q22;q22)] is among the most common recurrent cytogenetic abnormalities associated with acute myeloid leukemia (AML), appearing in 8-15% of AML patients. The t(8;21) results in a fusion of the AML1 (RUNX1 ) locus on chromosome 21 with the ETO (RUNX1T1 ) locus on chromosome 8, creating the AML1-ETO (AE) fusion protein. The AE fusion contains the N-terminal AML1 runt homology domain and nearly the entire ETO protein. Therefore, AE RNA transcripts have the ETO 3' untranslated region (3'UTR). Recent evidence suggests that AE expression level is an important factor in t(8;21) leukemogenesis. Single cell qPCR of t(8;21) AML patient samples shows greatly increased expression of AE from t(8;21)+ leukemia stem cells at diagnosis versus t(8;21)+ hematopoietic stem cells during remission (Shima T, et al. Exp. Hematol. 2014). Furthermore, cells highly expressing the AE9a truncation are required to establish disease in the AE9a transplantation mouse model (Link KA, et al. PNAS . 2016). Unpublished data from our lab indicates that the level of AE transcripts is substantially higher in t(8;21)+ leukemic blasts versus t(8;21)+ differentiated monocytes/granulocytes. More importantly, this enhanced expression is associated with shortening of the AE 3'UTR. Therefore, identifying the factors that control AE expression via its 3'UTR may provide valuable insight into t(8;21) leukemogenesis. Post-transcriptional regulation of mRNA occurs primarily through sequence specific interactions between cis -elements in the 3'UTR and trans -factors, such as micro-RNAs (miRNA). We sought to determine the specific AE 3'UTR cis -elements and trans -factors involved in the regulation of AE expression.The full-length AE 3'UTR is 5.2kb long. However, we detected several shorter-length isoforms in t(8;21) patients samples. To determine the effect on expression contributed by these 3'UTR isoforms, we made a series of luciferase reporters containing the different AE 3'UTR isoform sequences. In transient transfection assays in the t(8;21)+ Kasumi-1 and SKNO-1 cell lines, we observed an up to 5-fold difference in luciferase activities between the AE 3'UTR isoforms, with shorter 3'UTR isoforms having significantly higher activity. This result suggests that cis -elements within the longer AE 3'UTRs negatively affect AE expression. To characterize the complete set of cis -regulatory elements within the 5.2kb AE 3'UTR, we made luciferase reporters with a series of progressive 600bp fragments of the AE 3'UTR. Luciferase assays with this set of reporters in two t(8;21) AML cell lines Kasumi-1 and SKNO-1 showed significant differences in luciferase activity between the fragments (up to 5-fold). Interestingly, both t(8;21)+ cell lines had a similar pattern of luciferase activity, with a consistently down-regulated region that is only present in longer AE 3'UTR isoforms. Furthermore, comparing to results in non-t(8;21) cell lines (K562, U937, 293T, HL-60, and THP-1), we found t(8;21)+ cell type specific patterns in regulation.We further sought to elucidate the role of miRNAs in regulating AE expression via the 3'UTR. Normal miRNA biogenesis requires a multi-step process involving the RNAase DICER. To assess the contribution of miRNAs in AE expression, we inhibited miRNA biogenesis by knocking down DICER in the t(8;21)+ Kasumi-1 and SKNO-1 cell lines. These cells showed increased AE expression by qPCR and western-blot, compared to controls. Furthermore, DICER knockdown significantly increased luciferase reporter activity in a subset of the 600bp AE 3'UTR fragments corresponding to the down-regulated region of the 3'UTR. Together these data suggest that there is a miRNA targeted regulatory region that is only present in longer AE 3'UTR isoforms. This supports a model where shortened 3'UTRs avoid miRNA targeted down-regulation by eliminating their binding sites from AE transcripts to increase the fusion gene expression.Collectively, we examined the influence of AE 3'UTR isoforms on expression, mapped the regulatory elements within the AE 3'UTR, and identified the region that is involved in miRNA mediated regulation. Combining these results with miRNA target prediction programs and publicly available t(8;21) patient miRNA expression data, we have generated a list of potential miRNAs that target this region and are examining their role in regulating AE expression. DisclosuresNo relevant conflicts of interest to declare.

ETO2 Regulates Cell-Fate Genes and Controls Relapse in Acute Myeloid Leukemia

The cell-fate of hematopoietic stem cells (HSCs) and their malignant version, leukemia stem cells (LSCs), are controlled by antagonistic functions of two classes of genes. We defined these genes as pro-HSC/LSC and anti-HSC/LSC genes based on their abilities to increase or decrease the steady state numbers of stem cells. Expansion of LSCs is thought to be an early step towards relapse (Lapidot, Sirard et al. 1994, Huntly and Gilliland 2005, Vasanthakumar and Godley 2014), a medical condition with no effective cures. We show here that the expression of both pro- and anti-HSC/LSC genes are coordinately controlled by ETO2, a transcriptional corepressor of E-protein family of transcription factors (Zhang, Kalkum et al. 2004, Gow, Guo et al. 2014). In both HSCs and acute myeloid leukemia (AML) cells, ETO2 occupies active chromatin regions that encompass enhancers and promoters of pro- and anti-HSC/LSC genes. ETO2 orchestrates the binding of epigenetic modifiers and transcription factors to enhance the expression of self-renewal and survival genes while reducing the expression of growth arrest and pro-inflammatory genes. Further supporting a role of ETO2 in maintaining the self-renewable population of HSCs and LSCs, depleting ETO2 compromises self-renewal of HSCs and diminishes in vivo propagation of AML cells.Analysis of multiple patient datasets supports the conclusion that relapse requires a threshold level of ETO2 expression. Attaining this threshold level requires a conserved gene activation mechanism mediated by GCN5 histone acetyltransferase. Inhibiting GCN5 reduces ETO2 and abolishes colony-formation capacity of primary AML cells. We also show that the conserved and diverged functions between ETO2 and AML1-ETO, a leukemia fusion protein expressed in t(8;21) AML, combined with a newly-discovered ability of AML1-ETO to repress ETO2 transcription, defines the unique properties of t(8;21) AML and explains its long-observed favorable prognosis. Importantly, we show that despite an enriched expression of HSC/LSC genes, relapse is paused in t(8;21) AML due to AML1-ETO-mediated downregulation of ETO2 and requires overcoming this repression to proceed. Thus, targeting GCN5/ETO2 axis to achieve a similar effect as that imposed by AML1-ETO is expected to dramatically improve therapeutic efficacy of AML, whose current survival rate is worse than most cancers and leukemias (Supported by NIH grants R01HL093195-01A1 and R21CA178513 to JZ and T32 training grant T32GM008306-26A1 to NS).Gow, C. H., C. Guo, D. Wang, Q. Hu and J. Zhang (2014). “Differential involvement of E2A-corepressor interactions in distinct leukemogenic pathways.” Nucleic Acids Res 42(1): 137-152.Huntly, B. J. and D. G. Gilliland (2005). “Leukaemia stem cells and the evolution of cancer-stem-cell research.” Nat Rev Cancer 5(4): 311-321.Lapidot, T., C. Sirard, J. Vormoor, B. Murdoch, T. Hoang, J. Caceres-Cortes, M. Minden, B. Paterson, M. A. Caligiuri and J. E. Dick (1994). “A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.” Nature 367(6464): 645-648.Vasanthakumar, A. and L. A. Godley (2014). “On the origin of leukemic species.” Cell Stem Cell 14(4): 421-422.Zhang, J., M. Kalkum, S. Yamamura, B. T. Chait and R. G. Roeder (2004). “E protein silencing by the leukemogenic AML1-ETO fusion protein.” Science 305(5688): 1286-1289. DisclosuresNo relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Mimic BET Inhibitors and Cause RNA Polymerase Pausing at MYC, KIT, and Other Oncogenes in t(8;21) AML

AML containing the t(8;21) is exceptionally sensitive to the FDA approved HDAC inhibitors. While these compounds cause extensive, S phase-dependent DNA damage by stalling replication forks, they also activate transcription by increasing acetylation of the N-terminal histone tails that are “read” by chromatin modifying enzymes. However, gene expression analysis showed that these compounds turn off many genes even though histone acetylation is commonly associated with gene activation. To address this conundrum, we performed precision nuclear run-on transcription analysis (PRO-seq) of t(8;21)-expressing cells treated with the class I HDAC inhibitors depsipeptide, vorinostat, and panobinostat and the HDAC3-selective compound RPG966 for 1 or 4 hr and found that the broad-spectrum compounds activated a relatively small number of genes while causing RNA polymerase pausing at key oncogenic loci including MYC, KIT, BCL-XL, and BCL6 . By contrast, RGFP966 mostly activated gene expression, which appeared to be associated with increased initiation. In our previous study (Zhao et al, Cell Reports, 2016) we reported genes with RNA polymerase pausing at promoters after bromodomain and extra terminal (BET) inhibitor treatment. The majority of those sites also displayed RNA polymerase pausing after treatment with broad-spectrum HDAC inhibitors. Analysis of histone modifications by western blot and ChIP-seq found large increases in marks bound by BRD4 and other BET family members. ChIP-exo and ChIP-seq also found a loss of proper localization by BRD4 after HDAC inhibitor treatment. Thus, HDAC inhibitors massive increases in global histone acetylation, which triggers “epigenetic confusion”, whereby BRD4 cannot locate its normal chromatin address in high levels of nucleosome acetylation. Therefore, for genes regulated by BRD4, HDAC inhibitors act similarly to BET inhibitors, but can also cause DNA damage to yield a potent combination mechanism of action against t(8;21) AML. Interestingly, almost half of AML1-ETO fusion protein target genes also had change in promoter proximal pausing of RNA polymerase after Depsi, SAHA or RGFP966 treatment, suggesting that HDAC inhibitors may more directly perturb the effects of AML1-ETO. DisclosuresNo relevant conflicts of interest to declare.

Repurposing cabozantinib with therapeutic potential in KIT-driven t(8;21) acute myeloid leukaemias

Cabozantinib is an orally available, multi-target tyrosine kinase inhibitor approved for the treatment of several solid tumours and known to inhibit KIT tyrosine kinase. In acute myeloid leukaemia (AML), aberrant KIT tyrosine kinase often coexists with t(8;21) to drive leukaemogenesis. Here we evaluated the potential therapeutic effect of cabozantinib on a selected AML subtype characterised by t(8;21) coupled with KIT mutation. Cabozantinib exerted substantial cytotoxicity in Kasumi-1 cells with an IC50 of 88.06 ± 4.32 nM, which was well within clinically achievable plasma levels. The suppression of KIT phosphorylation and its downstream signals, including AKT/mTOR, STAT3, and ERK1/2, was elicited by cabozantinib treatment and associated with subsequent alterations of cell cycle- and apoptosis-related molecules. Cabozantinib also disrupted the synthesis of an AML1-ETO fusion protein in a dose- and time-dependent manner. In a mouse xenograft model, cabozantinib suppressed tumourigenesis at 10 mg/kg and significantly prolonged survival of the mice. Further RNA-sequencing analysis revealed that mTOR-mediated signalling pathways were substantially inactivated by cabozantinib treatment, causing the downregulation of ribosome biogenesis and glycolysis, along with myeloid leukocyte activation. We suggest that cabozantinib may be effective in the treatment of AML with t(8;21) and KIT mutation. Relevant clinical trials are warranted.

Histone deacetylase 3 preferentially binds and collaborates with the transcription factor RUNX1 to repress AML1–ETO–dependent transcription in t(8;21) AML

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.

Oncogenic Roles Of A Histone Methyltransferase SETDB2 In AML1-ETO Positive AML.

IntroductionAML1-ETO produced by t(8;21) abnomality has multiple effects on the leukemogenesis of acute myeloid leukemia (AML). SET domain, bifurcated 2 (SETDB2) can mediate gene silencing by trimethylation of the ninth lysine residue of histone H3 protein (H3K9) of the promoter and has been confirmed as an oncogene in many cancers. The role of SETDB2 in AML1-ETO positive AML is not clear.MethodsQuantitative reverse transcription PCR was performed to measure SETDB2 expression in bone marrow from AML patients and healthy people. Kaplan-Meier analysis was performed to investigate the effect of SETDB2 on prognosis of AML patients. Dual luciferase reporter gene assay, chromatin immunoprecipitation were performed to investigate the regulatory mechanism of AML1-ETO on SETDB2. CCK-8 and colony formation assay were performed to measure the effect of SETDB2 on leukemic cells.ResultsSETDB2 is highly expressed in AML1-ETO positive AML. The overall survival, event-free and relapse-free survival rate of patients with high SETDB2 expression was lower than those of patients with low SETDB2 expression. SETDB2 is epigenetically upregulated by AML1-ETO fusion protein. Downregulation of SETDB2 expression significantly inhibits the proliferation and clonality of leukemic cells and promotes the sensitivity of leukemic cells to an epigenetic inhibitor JQ1.ConclusionAML1-ETO/SETDB2 is a novel epigenetic pathway of leukemogenesis and SETDB2 is a potential therapeutic target of t(8;21) AML.

The acute myeloid leukemia associated AML1-ETO fusion protein alters the transcriptome and cellular progression in a single-oncogene expressing in vitro induced pluripotent stem cell based granulocyte differentiation model.

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease.

Peripheral Blood Absolute Lymphocyte Count at Diagnosis As a New Prognostic Factor in Acute Myeloid Leukemia with t(8;21) Patients

Background: The recurrent translocation t(8;21) represents approximately 12% of adult AML cases, and it involves the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, which generates an AML1-ETO fusion protein. Previous studies have shown that t(8;21) AML with AML1-ETO fusion has a high complete remission rate with standard chemotherapy and a prolonged survival when sequential high-dose cytarabine is administered. However, prognosis of AML with t(8;21) is heterogeneous. Method: In order to under the relationship between clinical characteristics and prognosis, we retrospectively analyzed data of 68 t(8;21) AML patients who diagnosed at the two centers of South Korea between November 2005 and September 2016. Results: Median age was 56 (range, 17 to 82) and median follow up duration was 99.8 months (range, 278 to 150.8). Among 65 patients who achieved CR after induction therapy, 24 patients (37%) received allogeneic stem cell transplantation (allo-SCT). The median absolute lymphocyte count (ALC) at diagnosis was 2,093 × 106/L (range, 400-7,200), with higher ALC (≥ 2,205 x 106/L) being detected in 31 (45.6%) patients. Higher ALC at diagnosis was defined by an ALC cut-off of ≥ 2,205 x 106/L determined by receiver operating characteristic analysis and this cutoff value discriminated (in terms of sensitivity and specificity) between survival and death (area under curve [AUC] = 0.740, P = 0.001). In univariate analysis, lower CD19 expression (≥ 35%), higher BM blast (≥ 55%) and higher ALC at diagnosis were significantly associated with poor overall survival (OS) and leukemia-free survival (LFS) rate. The 5-year OS and the 5-year LFS of patients with higher ALC were 26.2% and 26.7%, respectively, while those of patients with lower ALC were 83.9 % and 73.6%, respectively. Of note, when we analyzed OS of 139 patients with normal karyotype according to ALC, there was not significant difference of survival rate and the 5-year OS of higher ALC in normal karyotype AML patients was 21.2%. Both groups according to ALC in t(8;21) AML patients were comparable for age, sex and clinical features at diagnosis as well as the proportion of patients who received allo-SCT. WBC count (&gt; 20,000 x 109/L), ANC (&gt; 1,500 x 106/L), additional cytogenetic abnormality, and C-kit mutation at diagnosis have not significant impact on survival. Minimal residual disease (MRD) after the first consolidation therapy did not affect the survival rate. Interestingly, among the patients who achieved CR after induction therapy, patients in higher ALC group showed MRD after the first consolidation therapy with higher incidence (91.3% vs. 60.6%, P = 0.012). In multivariate analysis, higher BM blast (HR 3.97, P = 0.004) and higher ALC (HR 11.59, P &lt; 0.001) remained independent prognostic factors for poor OS rates. Higher BM blast (HR 4.42, P = 0.001) and higher ALC (HR 7.37, P &lt; 0.001) were also independent factors for worse LFS rates. Conclusion: Higher ALC was a significant prognostic factor for LFS and OS in AML patients with t(8;21). Further study for the correlation of ALC and the mechanism of adverse prognosis are needed. Disclosures No relevant conflicts of interest to declare.

Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

The oncogenic fusion protein AML1-ETO retains the ability of AML1 to interact with the enhancer core DNA sequences, but blocks AML1-dependent transcription. Previous studies have shown that post-translational modification of AML1-ETO may play a role in its regulation. Here we report that AML1-ETO-positive patients, with high histone lysine methyltransferase Enhancer of zeste homolog 1 (EZH1) expression, show a worse overall survival than those with lower EZH1 expression. EZH1 knockdown impairs survival and proliferation of AML1-ETO-expressing cells in vitro and in vivo. We find that EZH1 WD domain binds to the AML1-ETO NHR1 domain and methylates AML1-ETO at lysine 43 (Lys43). This requires the EZH1 SET domain, which augments AML1-ETO-dependent repression of tumor suppressor genes. Loss of Lys43 methylation by point mutation or domain deletion impairs AML1-ETO-repressive activity. These findings highlight the role of EZH1 in non-histone lysine methylation, indicating that cooperation between AML1-ETO and EZH1 and AML1-ETO site-specific lysine methylation promote AML1-ETO transcriptional repression in leukemia.

Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO-containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.

Role of AML1-ETO Related Fusion Circular RNA in the Growth of Leukemia Cells

Introduction: The pathogenesis of acute myeloid leukemia (AML) is always associated with chromosomal translocation, such as t(8;21)(q21;q22), which results in the formation of AML1-ETOfusion gene. The AML1-ETO fusion protein leads to hematopoietic differentiation blocked and leukemogenesis. How genomic abnormities such as chromosomal translocation influence the production and biological function of noncoding RNAs is relatively less studied. With the application of high-throughput sequencing and bioinformatics, it is increasing clear that the circular RNAs (circRNA) played important role in multiple biological processes. However, circRNA is rarely studied in hematopoietic malignancies. Here we assessed the roles of AML1-ETO related fusion circRNA (F-CircAE) in AML1-ETO leukemia.Methods: Total RNA was extracted from leukemia cell lines Kasumi-1 and SKNO-1, then treated with RNaseR or not. PCR assay with divergent primers was performed to identify F-CircAEs. The F-CircAE retroviral expression vector was constructed using Gibson Assembly Cloning Kit. NIH3T3 cells were transduced with retrovirus, the proliferation and foci formation were detected by crystal violet staining. F-CircAE-expressingNIH3T3 cells were injected subcutaneously into nude mice. Tumor growth was monitored at indicated time. To knock down the expression of F-CircAEs, the short hairpin double-stranded oligo targeting the back-splice junction of the F-CircAEs was inserted into the pLKO.1 lentiviral vector. Kasumi-1 cells were transduced with lentivirus, then the proliferation and cell cycle distribution of Kasumi-1 cells was measured by MTS assay and PI staining. Western blot was used to detect the cell cycle associated protein expression. RNA pulldown assay was performed with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the instructions. The bound proteins in the pulldown assay were analyzed by spectrum analysis. RNA-seq were performed using the Illumina platform. Sequencing reads were analyzed by a standard RNA-Seq pipeline. Gene Set Enrichment Analysis (GSEA) were used to analyze the pathway enrichment.Results: Several F-CircAEswere identified in AML1-ETO leukemia cell lines (Kasumi-1, SKNO-1) and t(8;21) patients' bone marrow mononuclear cells (BMMCs), but not in K562 cells and donors' BMMCs (Figure 1). To further validate F-CircAEs, RNaseR treatment was conducted and amplification products of circular RNAs were still detectable in AML1-ETO positive cells (Figure 1). Subsequently, Sanger sequencing of these amplification circular products revealed the head to tail sites of F-CircAE.To examine the function of F-CircAE, overexprseeion of F-CircAE in NIH3T3 cells resulted in increased proliferation compared with mutated F-CircAE (F-CircAE-mut) overexprssion or control group (Figure 2). Meanwhile, more foci were formed in the overexpressed F-CircAE group. In vivo,F-CircAE overexpression also promoted the growth of NIH3T3 cells in the injection site of nude mice. Furthermore, silencing of two F-CircAEs reduced the growth rates of Kasumi-1 cells (Figure 3) and induced an increase in G1 phase and a decrease in S and G2/M phase compared with scramble group. The protein levels of CDK2 and CyclinD1, which were the cell cycle regulatory components at G1 boundary, were reduced in F-CircAE knockdown groups compared with scramble group. To explore the mechanism by which F-CircAE promoted leukemia cells proliferation, RNA-pulldown assay was performed and more than 70 proteins were found to bind to F-CircAE. Based on the sequencing reads of RNA-seq, GSEA revealed several pathways were enriched following F-CircAE knockdown in Kasumi-1 cells.Conclusions: F-CircAEs were existed in t (8;21) leukemia cell lines and primary AML leukemia patients' BMMCs. F-CircAE could bind to proteins involved in the growth of leukemia cells. Discovery of F-CircAEs in AML1-ETO leukemia could make an immense progress in understanding their pathogenic mechanism, indicating new marker for diagnosis and therapy target. DisclosuresNo relevant conflicts of interest to declare.

The Three E Proteins Define a Heterogeneity of the AML1-ETO-Containing Transcription Factor Complex (AETFC) and Differentially Regulate t(8;21) Leukemogenesis

The leukemogenic AML1-ETO fusion protein is produced by the t(8;21) translocation, which is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AETFC, that contains multiple transcription factors and cofactors. Among these AETFC components, E2A (also known as TCF3) and HEB (also known as TCF12), two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA (E-box) binding capacity to AETFC, and are functionally essential for leukemogenesis. However, we find that the third E protein, E2-2 (also known as TCF4), is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the basic helix-loop-helix (bHLH) DNA-binding domain of E2-2. Gene expression profiling and ChIP-seq analysis reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, consistent with the fact that E2-2 is a critical transcription factor in dendritic cell (DC) development, our studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with DC differentiation, and that restoration of E2-2 triggers a partial differentiation of the AML1-ETO-expressing leukemic cells into the DC lineage. Meanwhile, E2-2, but not E2A or HEB, represses MYC target genes, which may also contribute to leukemic cell differentiation and apoptosis. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates the development of leukemia. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a molecular heterogeneity of AETFC, which merits further study in different t(8;21) AML patients, as well as in its potential regulation of cellular heterogeneity of AML. These studies should improve our understanding of the precise mechanism of leukemogenesis and assist development of diagnostic and therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.