AML1-ETO Fusion Research Articles

Frequency and clinical features of ASXL2 gene mutation in acute myeloid leukemia patients with AML1- ETO fusion gene positive

目的探讨伴AML1-ETO融合基因的急性髓系白血病（AML）患者ASXL2基因突变情况、突变阳性患者临床特征及ASXL2基因突变与c-kit基因突变的关系。方法采用PCR扩增产物片段直接测序分析法，检测59例伴AML1-ETO融合基因初发AML患者ASXL2基因第11、12外显子编码区突变情况，比较ASXL2基因突变阳性和阴性组患者的临床特征、生存及c-kit基因突变情况。结果59例患者中7例存在ASXL2突变，突变率为11.9%。ASXL2基因突变阳性组患者初诊时外周血红蛋白浓度中位数为56.2（38.0~72.0）g/L，显著低于ASXL2突变阴性组患者的69.0（37.2~154.0）g/L，差异有统计学意义（P=0.038）；外周血WBC、PLT、嗜酸粒细胞比例、骨髓原始细胞比例与ASXL2突变阴性组相比，差异均无统计学意义（P值均>0.05）。两组均未见肝、脾、中枢神经系统浸润；淋巴结不同程度肿大，但ASXL2基因突变阳性、阴性两组间差异无统计学意义（P=0.859）。免疫表型分析显示：ASXL2基因突变阳性组CD33表达显著低于阴性组（P=0.033）；两组患者均未表达cCD3，CD117、cMPO、HLA-DR、CD34、CD38、CD13、CD44、CD15、CD64、CD11b、CD56、CD19、cCD79a、CD7两组表达差异均无统计学意义（P值均>0.05）。ASXL2基因突变阳性与阴性组患者总缓解率、总生存时间差异均无统计学意义（P值分别为0.577、0.631）。两组c-kit基因突变检出率分别为14.3％和29.4%，差异无统计学意义（P=0.697）。结论该组伴AML1-ETO融合基因AML患者ASXL2基因突变率为11.9%。ASXL2突变阳性患者外周血红蛋白浓度、CD33表达方面呈现一定的临床特征。ASXL2基因突变与c-kit基因变突可能没有特定的关联性。

Salicylate, diflunisal and their metabolites inhibit CBP/p300 and exhibit anticancer activity.

Salicylate and acetylsalicylic acid are potent and widely used anti-inflammatory drugs. They are thought to exert their therapeutic effects through multiple mechanisms, including the inhibition of cyclo-oxygenases, modulation of NF-κB activity, and direct activation of AMPK. However, the full spectrum of their activities is incompletely understood. Here we show that salicylate specifically inhibits CBP and p300 lysine acetyltransferase activity in vitro by direct competition with acetyl-Coenzyme A at the catalytic site. We used a chemical structure-similarity search to identify another anti-inflammatory drug, diflunisal, that inhibits p300 more potently than salicylate. At concentrations attainable in human plasma after oral administration, both salicylate and diflunisal blocked the acetylation of lysine residues on histone and non-histone proteins in cells. Finally, we found that diflunisal suppressed the growth of p300-dependent leukemia cell lines expressing AML1-ETO fusion protein in vitro and in vivo. These results highlight a novel epigenetic regulatory mechanism of action for salicylate and derivative drugs.

Research progress of histon deacetylase inhibitors in AML1-ETO positive acute myeloid leukemia

The t(8; 21) (q22; q22) results in fusion of acute myeloid leukemia (AML)1 gene on chromosome 21 and ETO gene on chromosome 8, and produces AML1-ETO fusion gene, which plays a critical role in leukemogenesis by recruiting mSin3/N-CoR/SMART/HDACs corepressor complex to promoter of AML1 target genes including interluekin (IL)-3 and granulocyte macrophage-colony stimulating factor (GM-CSF) related to differentiation of hematopoietic stem cells and inactivating tumor suppressor genes. Histon deacetylase inhibitors (HDACI) reexpress silenced suppressor genes through regulation of histone acetylation and chromatin remodeling. Moreover, some of HDACI are able to degrade AML1-ETO fusion and play a selective role in AML1-ETO positive AML. The combinations of HDACI with DNA hypomethylated medicine, glucocorticoid, heat shock protein (HSP)-90 inhibitor and arsenic trioxide (ATO) are explored for the therapy of AML1-ETO positive AML. Herein, we discuss the effect and mechanism of HDACI on fusion protein of the AML1-ETO, and its target genes, as well as the mechanism in treatment of AML1-ETO positive AML with HDACI. Key words: Histone deacetylase inhibitors; Core binding factors; Leukemia, myeloid, acute

Celastrol Induces Cell Apoptosis and Inhibits the Expression of the AML1-ETO/C-KIT Oncoprotein in t(8;21) Leukemia.

Resistance to chemotherapy is a major challenge to improving overall survival in Acute Myeloid Leukemia (AML). Therefore, the development of innovative therapies and the identification of more novel agents for AML are urgently needed. Celastrol, a compound extracted from the Chinese herb Tripterygium wilfordii Hook, exerts anticancer activity. We investigated the effect of celastrol in the t(8;21) AML cell lines Kasumi-1 and SKNO-1. We demonstrated that inhibition of cell proliferation activated caspases and disrupted mitochondrial function. In addition, we found that celastrol downregulated the AML1-ETO fusion protein, therefore downregulating C-KIT kinases and inhibiting AKT, STAT3 and Erk1/2. These findings provide clear evidence that celastrol might provide clinical benefits to patients with t(8;21) leukemia.

Co-existence of AML1-ETO and BCR-ABL1 transcripts in a relapsed patient of acute myeloid leukemia with favorable risk group: A coincidence or clonal evolution?

Prognosis of acute myeloid leukemia relies heavily on the cytogenetic and molecular abnormalities. AML1-ETO fusion protein resulting from t(8;21), a recurring cytogenetic abnormality, is known to be associated with favorable prognosis. Additional molecular defects may, however, co-operate with the fusion proteins and alter the course of the disease. Among the additional cytogenetic defects, presence of Philadelphia (Ph) chromosome has rarely been documented in this subtype. Little is known about the consequences of its interactions with AML1-ETO, and its effect on morphological and clinical picture. Moreover, Ph+ clones or subclones may appear at any point during the disease course. We herein report one such unusual case of a 26-year-old female, who was diagnosed to have t(8;21) and managed accordingly. During disease relapse after 2.5years, the bone marrow showed extensive eosinophilia and basophilia. Subsequent molecular testing showed the presence of BCR-ABL in addition to the AML1-ETO fusion product.

The Homeobox Gene VENTX Collaborates with AML1-ETO in Inducing an Acute Leukemia in Mice

Homeobox genes are key factors in the development of acute leukemias. So far, little is known about the role of non-clustered homeobox genes such as VENTX. The Vent-like homeobox gene VENTX is a member of the Vent gene family in mammals and is the mammalian homolog of the Xenopus xvent gene. Our group has previously shown that VENTX promotes myeloid differentiation when overexpressed in normal CD34+ human stem - and progenitor cells and shows high and aberrant expression in human acute myeloid leukemia (AML) characterized by the AML1-ETO (AE) fusion (Rawat et al., PNAS 2010). To prove the functional relevance of aberrant VENTX expression in this AML genotype, we set up in vitro assays and a murine bone marrow transplantation model, mimicking high expression of VENTX by retrovirally engineered expression of the gene in murine bone marrow progenitor cells. In the CFU-S assay coexpression of both genes significantly increased the frequency of spleen colonies 14-fold and 7-fold compared to VENTX and AE, respectively (p<0.02), and more than 7-fold compared to the control (p<0.001). Most importantly and in contrast to AE alone, coexpression of AE and VENTX induced an acute leukemia after a median latency of 11.3 months post transplant. All leukemias evaluated were rapidly transplantable with a median latency until leukemia induced death of 40 days. The median blast percentage in primary recipient mice was 66% (range 35-100%, n=24). Of note, massive expansion of the red pulpa was documented with the appearance of erythroblasts in the spleen. Furthermore, blasts in the bone marrow expressed CD19, partly in combination with Sca1 and Gr1, pointing to an aberrant expression of this antigen in the AE/VENTX leukemias. Ex vivo, leukemic cells grew permanently generating AE/VENTX positive cell lines. RNA-Seq analyses from CD34+ cord blood transduced with VENTX documented 279 differentially expressed genes compared to the GFP control, hitting 7 pathways (Hematopoietic cell lineage, Cytokine-cytokine receptor pathway, Jak-STAT signaling pathway, Porphyrin and Chlorophyll Metabolism, Asthma, Thyroid cancer and Endocytosis) in the KEGG-Analysis and showed a downregulation of genes necessary for terminal erythroid differentiation such as the EPO-receptor and GATA1. Taken together, these data characterize VENTX as an important contributing factor to AE positive leukemias. Importantly, coexpression of AE and VENTX allows the generation of a murine AE model, which in contrast to all other published mouse models mimics aberrant expression of CD19 in human AE positive AML, a hallmark of this AML genotype. DisclosuresBuske:CELLTRION, Inc.: Consultancy, Honoraria.

UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in human preleukemia induced by AML1-ETO.

The t(8;21) rearrangement, which creates the AML1-ETO fusion protein, represents the most common chromosomal translocation in acute myeloid leukemia (AML). Clinical data suggest that CBL mutations are a frequent event in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been investigated. In this study, we demonstrate that CBL mutations collaborate with AML1-ETO to expand human CD34+ cells both in vitro and in a xenograft model. CBL depletion by shRNA also promotes the growth of AML1-ETO cells, demonstrating the inhibitory function of endogenous CBL in t(8;21) AML. Mechanistically, loss of CBL function confers hyper-responsiveness to thrombopoietin and enhances STAT5/AKT/ERK/Src signaling in AML1-ETO cells. Interestingly, we found the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, is upregulated by AML1-ETO through transcriptional and miR-9-mediated regulation. UBASH3B/Sts-1 depletion induces an aberrant pattern of CBL phosphorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused by UBASH3B/Sts-1 depletion can be rescued by ectopic expression of CBL mutants, suggesting that UBASH3B/Sts-1 supports the growth of AML1-ETO cells partly through modulation of CBL function. Our study reveals a role of CBL in restricting myeloid proliferation of human AML1-ETO-induced leukemia, and identifies UBASH3B/Sts-1 as a potential target for pharmaceutical intervention.

Abstract 3972: MicroRNAs as potential therapeutic agents for AML: Targeting the AML1-ETO Oncogene by pre-miR-520 and -373

Abstract Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by the accumulation of poorly differentiated myeloid cells in the bone marrow and blood of patients. Differentiation therapy is an attractive therapeutic approach for treating patients with AML. All-Trans Retionic Acid, induces differentiation of patients with the PML/RARa oncogene, but is ineffective in treating other more frequent types of AML. Here we describe the use of microRNAs to target the oncogene formed as a result of the (8;21) translocation, The goals was to identify miRNAs that could target the breakpoint region of the fusion protein, and determine if this would reduce expression of AML1-ETO and promote differentiation and/or apoptosis. Bioinformatics analysis was used to identify 28 different miRNAs that could potentially target AML1-ETO transcripts. Among those, miRNA-520 and -373 showed the highest degree of complimentary to AML1-ETO transcripts at the braikpoint. To look for miR-520 and -373 efficacy, we transfected two AML cell lines, Kasumi 1 and SKNO1, which have AML1-ETO translocation, with pre-miR-520 and -373 LNA probes, as well as HL60 cells which lack AML1-ETO. Expression of the LNA pre-miR-520/373 in Kasumi-1 and SKNO-1 cell lines decreased AML1-ETO transcripts and led to a significant reduction of AML1-ETO protein levels. The inhibition of AML1-ETO fusion protein also induced apoptosis of leukemic cells in vitro, but had no effect on HL60 cells. Moreover, the administration of LNA pre-miR-520 in an AML xenografts murine model increased apoptosis of leukemic cells and reduced tumor burden without obvious toxicity. We are currently testing the effects of miR-520 and -373 on differentiation and proliferation of primary blasts from patients with the AML1-ETO translocation. Our results suggest that small molecules such as miRNAs can be identified that directly target a frequent AML oncogene. miRNAs are naturally accruing molecules and have the potential advantage of producing modest side effects, with highly significant specificity. The approaches we developed in this study can be used to evaluate and optimize other pre-miRs and anti-miRs as therapeutic agents to target other types oncogenes not currently amenable to small molecule drugs. Citation Format: Patricia A. Toniolo, Patrick M. Pilarski, Christian Bach, James D. Griffin, Sophia Adamia. MicroRNAs as potential therapeutic agents for AML: Targeting the AML1-ETO Oncogene by pre-miR-520 and -373. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2015-3972

Clinical significances of WT1 gene and AML1-ETO fusion gene expression for monitoring minimal residual disease in children with acute myeloid leukemia

Objective To explore the diagnostic value of WT1 gene and AML1-ETO fusion gene in the monitoring of minimal residual disease (MRD) and prediction of relapse in childhood acute myeloid leukemia (AML). Methods From May 2007 to March 2015, a total of 13 AML1-ETO fusion gene positive AML children and 15 leukemia fusion gene negative AML children who received initial treatment in Department of Pediatrics in Sichuan Provincial People's Hospital were enrolled as study objects, and divided into fusion gene positive group (n=13) and fusion gene negative group (n=15). Relative expression levels of WT1 gene and AML1-ETO fusion gene were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technique in bone marrow samples from children of two groups at the beginning of treatment and during follow-up period. Correlation of relative expression levels of WT1 gene and AML1-ETO fusion gene were analyzed by SPSS statistical software. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio of MRD predicting relapse were calculated and compared. Kaplan-Meier method was used to compare the relapse-free survival (RFS) rate between two groups and to assess MRD on the prediction of relapse. The study protocol was approved by the Ethical Review Board of investigation in Human Being of Sichuan Provincial People's Hospital.Informed consent was obtained from all participants. Results ①The relative expression levels of WT1 gene and AML1-ETO fusion gene were positively correlated in 83 bone marrow samples from 13 cases of fusion gene positive group (R2=0.874, P 0.05) at the time points of first diagnosis, first induction of remission, and relapse. ③After the first time induction of remission in the fusion gene positive group children, AML1-ETO quantitative levels as predictive relapse of diagnostic indicators were the highest in the sensitivity, accuracy, positive predictive value, negative predictive value as 85.7%, 76.9%, 75.0% and 80.0% respectively and negative likelihood ratio was minimum as 0.29. The specificity of WT1 gene in the fusion gene negative group was highest as 71.4%. ④After the first time induction of remission, there were 8 patients whose relative expression levels of AML1-ETO fusion gene were more than 0.01 in fusion gene positive group. Among these 8 cases, 6 patients relapsed and relapse rate was 75.0%. Relative expression levels of AML1-ETO fusion gene in other 5 cases were less than 0.01. Among the 5 cases, 1 patient relapsed and relapse rate was 20.0%. The difference of relapse rate between patents whose relative expression levels of AML1-ETO fusion gene were more and less than 0.01 was statistically significant (χ2=13.0, P=0.000). Up to the time of the follow-up , RFS rate of the patients with the relative expression level of AML1-ETO fusion gene more than 0.01 was significantly lower than that of patients with the relative expression level less than 0.01 (P=0.001). ⑤ After the first time induction of remission, there were 8 patients whose relative expression levels of WT1 gene were more than 0.01 in fusion gene negtive group. Among these 8 cases, 6 patients relapsed and relapse rate was 75.0%. Relative expression levels of WT1 gene in other 7 cases were less than 0.01. Among the 7 cases, 1 case relapsed and relapse rate was 14.3%. The difference of relapse rate between patients whose relative expression levels of WT1 gene were more and less than 0.01 was statistically significant (χ2=15.0, P=0.000). Up to the time of the follow-up , RFS rate of the patients with the relative expression level of WT1 gene more than 0.01 was significantly lower than that of patients with the relative expression level less than 0.01 (P=0.004). Conclusions AML1-ETO fusion gene is suitable for monitoring the MRD of AML children with fusion gene positive. WT1 gene is more suitable for detecting MRD and predicting relapse in AML children with fusion gene negative. It may indicate relapse when WT1 and AML1-ETO fusion gene relative expression levels significantly increased. Key words: Leukemia, myeloid, acute; gene, Wilms' tumor; AML1-ETO fusion gene, human; Neoplasm, residual; Polymerase chain reaction

The Transcriptome Study of Subtype M2 Acute Myeloblastic Leukemia.

Our objective is to explore the tumor-specific mutated genes by transcriptome sequencing of patients with acute myeloblastic leukemia. 96 patients with subtype M2 acute myeloid leukemia (AML), admitted during January 2007 to January 2012, were selected. Bone marrow and peripheral blood samples from the patients after the first visit and the patients who were improved or alleviated, were subjected to high-throughput sequencing to compare the gene expression. The single nucleotide mutation related to subtype M2 AML was detected. Meanwhile, real-time fluorescent quantitation RT-PCR was used to detect the AML1/ETO fusion gene and its correlation with prognosis after treatment. Among 96 patients, AML1-ETO fusion gene was positive in 52 cases, the positive rate was 54.17%. The complete relief (CR) rate of AML1-ETO fusion gene positive patients was 84.62%, and the CR rate of AML1/ETO fusion gene negative patients was 77.27%; the CR rate of AML1-ETO positive patients was higher than that of patients without the fusion gene, however there was no statistical difference. In the analysis of recurrent gene mutation in AML-M2 patients, IDH2, ASXL1, TET2, JAK1 and JAK2 gene expressions were not significantly different before treatment and after CR, however, IDHI, JAK3, ABL1 and BCR gene expressions were significantly different. In the study of transcriptome in AML-M2 patients, high-throughput sequencing could effectively detect the difference of the gene expression before treatment and after CR. Furthermore, positive expression of AML1-ETO fusion gene had effect on the prognosis of patients.

Identification of the Adapter Molecule MTSS1 as a Potential Oncogene-Specific Tumor Suppressor in Acute Myeloid Leukemia.

The adapter protein metastasis suppressor 1 (MTSS1) is implicated as a tumor suppressor or tumor promoter, depending on the type of solid cancer. Here, we identified Mtss1 expression to be increased in AML subsets with favorable outcome, while suppressed in high risk AML patients. High expression of MTSS1 predicted better clinical outcome of patients with normal-karyotype AML. Mechanistically, MTSS1 expression was negatively regulated by FLT3-ITD signaling but enhanced by the AML1-ETO fusion protein. DNMT3B, a negative regulator of MTSS1, showed strong binding to the MTSS1 promoter in PML-RARA positive but not AML1-ETO positive cells, suggesting that AML1-ETO leads to derepression of MTSS1. Pharmacological treatment of AML cell lines carrying the FLT3-ITD mutation with the specific FLT3 inhibitor PKC-412 caused upregulation of MTSS1. Moreover, treatment of acute promyelocytic cells (APL) with all-trans retinoic acid (ATRA) increased MTSS1 mRNA levels. Taken together, our findings suggest that MTSS1 might have a context-dependent function and could act as a tumor suppressor, which is pharmacologically targetable in AML patients.

Phenomic Screen in Vivo and in Vitro to Explore Novel Pathogenesis of AML1-ETO-Positive Leukemia Using PSC-Derived Hematopoietic Cells

Onset of acute myeloid leukemia (AML) has been accounted for by cooperation between multiple genetic alterations. Among the previously listed leukemogenic lesions, AML1-ETO fusion (AE) generated by translocation (8;21) (q22;q22) is one of the common mutations observed in 20-40% of patients. AE affects transcriptional regulation associated with hematopoietic differentiation, while 60% of AE-positive AML cases are shown to have together other types of mutation of genes involved in cell proliferation, such as receptor tyrosine kinase (RTK) c-kit and FLT3. Those data are compatible with so-called “multi-step leukemogenesis” model. At the same time, variety in clinical phenotypes even among patients harboring same sets of mutation strongly indicates the yet unknown “cooperative ” cues in genetics or epigenetics. From this viewpoint, finely elucidating “What kinds of such mechanisms” give “What kinds of impact associating with AE fusion” on “What kinds of target cells” during leukemogenesis is important.In order to address those problems in reproducible manner, we developed the novel phenomic screen system by combining reverse and forward genetic modifications with pluripotent stem cell (PSC)-derived hematopoietic culture. We induced the PiggyBac-based random knockdown motifs into PSC-derived hematopoietic cells that harbour artificial expression cassettes for AE fusion gene, then applied them for in vitro and in vivo assays. We performed these platform-based trials in parallel to the work with positive control cells carrying AE in addition to c-kit or Flt3-ITD mutations, recently identified poor prognosis conveyers among AE-positive AML. First, serial replanting assays in methylcellulose-containing semisolid media as well as liquid culture revealed that a few numbers of clones acquired significantly reinforced potential in cell growth and colony forming efficacy. As for positive control cells, they showed reproducibly strong tendency toward both increased colony forming efficacy and suppressed differentiation. Those results suggested the successful recapitulation of pathogenic cooperation between AE and RTK mutations in our PSC-derived hematopoietic cells, and confirmed the feasibility of our screening system. Interestingly, AE-positive myeloid lineage-committed progenitors as well as immature multipotent hematopoietic stem and progenitor cells (HSPCs) showed higher replating colony forming efficacy, which may indicate the representation of higher incidence of myeloblastic leukemia in AE-positive AML. We are identifying the knocked down genes and evaluating the perturbation of expression patterns of genes involved in differentiation, proliferation and survival as well as epigenetics associating genes within affected cells.In addition to in vitro assays, we next evaluated the feasibility of in vivo phenomic screening by performing intra bone marrow transplantation of gene modified PSC-derived progenitors into immunodeficient NOD/Shi-scid,IL-2RγKO Jic(NOG) mice. Also in these experiments, we successfully observed the cooperation between AE and RTK mutations for prolonged engraftment in case HSPCs and myeloid lineage-committed progenitors were transplanted. After that, we are now performing these screenings to select the phenotype acquiring clones.In conclusion, we successfully established a novel phenomic screen in vivo and in vitro to explore novel pathogenesis of AE-positive leukemia using PSC-derived hematopoietic cells. After confirming the recapitulation of already known cooperation between AE and RTK mutation, we are currently extending the screening using this platform. We believe that our model must allow us to better match treatment to prognosis across the disease spectrum via comprehensive understanding of pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

Tumor Suppressor FOXO1 Serves As a Critical Oncogenic Mediator in AML1-ETO Leukemia

AML1-ETO (AE) fusion protein, produced by the chromosome translocation (8;21), accounts for 10%-15% of acute myeloid leukemia (AML). We use CD34+ human umbilical cord blood (CB) cells retrovirally transduced with AE to model AE leukemia (AE cells). This model represents the pre-leukemic stage of AE leukemia since AE by itself does not lead to overt disease but largely enhances the self-renewal and impedes the differentiation of CB cells. However the molecular mechanism by which AE deregulates the hematopoietic program is still largely unknown.The FOXO subfamily of forkhead transcription factors, which in mammals includes FOXO1, FOXO3, FOXO4, and FOXO6, are well known tumor suppressor proteins due to their ability to arrest cell cycle and induce apoptosis. Although FOXO1 is silenced in many types of solid tumors, unexpectedly, we found expression of FOXO1 (but not other family members) significantly upregulated in AE AML patient samples compared to other AML subtypes. Increased protein expression was confirmed by western blot in a cohort of patient samples and our AE cells. AE bound to a region within intron 1 of the FOXO1 gene as shown by chromatin immunoprecipitation, the sequence of which includes several AML1 binding motifs. The expression of FOXO1 was dependent on the presence of AE as shown by overexpression and knockdown studies. These findings suggest that FOXO1 could be a direct target of AE. Knockdown of FOXO1 by shRNA resulted in decreased proliferation of AE cells, and conditional deletion of Foxo1 in murine AE cells led to decreased CFU replating in methylcellulose compared to non-deleted control. In contrast, conditional deletion of Foxo3 using the same approach did not significantly influence the clonogenicity of AE cells. These data indicate FOXO1 is critical for AE cell growth.Interestingly, overexpression of FOXO1 in CD34+ CB cells promoted their long-term proliferation in liquid culture, inhibited erythroid differentiation while promoting myeloid lineage development and enhanced their replating ability in colony forming assays. FOXO1-expressing cells also achieved a higher engraftment level when transplanted into immunodeficient mice. These effects partially phenocopy the outcomes observed upon AE expression in CB cells. These effects require the transcriptional activity of FOXO1, as shown with a FOXO1 mutant lacking DNA binding capacity, and are not seen upon FOXO3 expression. Although inhibition of reactive oxygen species (ROS) production is regarded as a general function of FOXO family proteins, ROS regulation is unlikely to be the major downstream mechanism, since ROS levels were only transiently and slightly decreased upon FOXO1 expression. In addition, treatment of CB cells with the ROS scavenger N-acetyl-cysteine could not recapitulate FOXO1’s phenotype.To gain molecular insight into FOXO1’s function in CB cells, RNA-Seq experiments were performed in triplicate. CD34+ CB cells were transduced with empty vector (MIT), FOXO1 DNA-binding-deficient mutant (FOXO1 DB), wildtype FOXO1 (FOXO1 WT) or AE. The MIT and FOXO1 DB groups showed very similar gene expression profiles. 1349 genes were upregulated and 1113 genes downregulated in the AE group while FOXO WT showed 608 genes increased and 490 genes decreased (P<=0.01, fold change>=1.5). To our surprise, 282 upregulated and 231 downregulated genes were common targets of AE and FOXO WT, suggesting that about 20% of AE downstream targets are potentially regulated via FOXO1. More strikingly, pathway enrichment analysis showed that published AE gene signature datasets were enriched in the FOXO1-WT gene list. Among genes upregulated by FOXO1, several have been reported as AE transcriptional targets, such as POU4F1 and JUP. In addition, some FOXO1 target genes, SOX4 and HLF for example, have been implicated in leukemogenesis.In summary, we find FOXO1 is required for AE cell growth, and overexpression of FOXO1 in CB cells can partially recapitulate the AE phenotype at both the morphological and molecular level. Taken together, these data suggest that instead of acting as a tumor suppressor, FOXO1 is a critical oncogenic mediator of the AE leukemia program. It is possible that FOXO1 serves as one signaling hub of the AE molecular network, similar to the role MEIS1-HOXA9 play in MLL-fusion AML. Fully dissecting the AE-FOXO1 pathway will enhance our understanding of AE leukemogenesis and provide potential therapeutic targets. DisclosuresMulloy:Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina.Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting.Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis.Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066).Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. DisclosuresNo relevant conflicts of interest to declare.

APP Gene Is Correlated with C-KIT Mutations and Indicates Poor Disease Outcome in AML1-ETO-Positive Acute Myeloid Leukemia

Amyloid precursor protein (APP) has been reported to be highly expressed in AML1-ETO-positive acute myeloid leukemia (AML1-ETO-positive AML), and we found it correlate with extramedullary infiltration regulated of by APP/ERK/MMP-2 signal pathway in our previous study. It is also known that C-KIT mutations highly expressed in AML1-ETO-positive AML and cooperates with full-length AML1-ETO to induce AML in mice. In this study we further described a close correlation of APP gene with C-KIT mutations, as well as APP related clinical and prognostic significance in 65 patients with AML1-ETO-positive AML.65 cases of AML1-ETO-positive AML patients with median age of 30 years old, who were admitted to our hospital from February, 2006 to June, 2013 and made the diagnosis according to WHO2008 diagnosis standard, were enrolled into this study. APP expression in bone marrow cells before the first chemotherapy was assessed by quantitative reverse transcriptase (QRT)-PCR method. These cases were accordingly divided into APP-H group (n=33, with high level of APP by QRT-PCR) and APP-L group (n=32, with lower level of APP by QRT-PCR) according to median APP expression. Incidence of C-KIT mutations, clinical characteristics and prognosis including complete response (CR), overall survival (OS), and recurrence free survival (RFS) with median 35 (6-96) months followed-up was differentiated between the two groups. Furthermore, expression of APP and AML1/ETO fusion gene were simultaneously monitored at the time of 3, 6, 12 and 24 months or relapse after CR by QRT-PCR method.The incidence of C-KIT mutations was significantly increased in the APP-H group, as compared with the APP-L group (39.4% versus 12.5%) and it was positively correlative with APP expression (rp=0.435, P=0.004). Of the 17 patients harboring C-KIT mutations, 13 patients overexpressed APP gene (P=0.014) (Figure 1). Clinically, APP-H patients exhibited significantly elevated white blood cells count, increased extramedullary infiltration (P=0.039 and P=0.019, respectively). Moreover, APP overexpression was related to low rate of two-cycle CR, RFS and OS (P=0.020, P=0.001 and P=0.029, respectively) (Table 1). In addition, the change of APP expression was consistent with that of AML1-ETO fusion gene monitored by QRT-PCR method at different status of leukemia, though APP expressed differently in different patients with the same AML1-ETO expression.Taken together, these data suggest that APP gene is correlated with C-KIT mutations and indicates poor disease outcome and dynamic monitoring APP expression could be another choice of minimal residual disease monitoring in AML1-ETO-positive AML. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RASSF2 Functions As a Tumor Suppressor in t(8;21) AML Via Promotion of Apoptosis

The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), found in approximately 12% of de novo AML cases. The majority of these cases are classified as FAB-subtype M2 AML. The t(8;21) results in the stable fusion of the AML1 (RUNX1) and ETO (RUNX1T1) genes. The AML1-ETO fusion protein is composed of the N-terminal portion of AML1, which includes the DNA-binding Runt-homology domain, and nearly the full-length ETO protein. The primary accepted mechanism by which AML1-ETO promotes leukemia development is through the aberrant recruitment of transcriptional repression/activation complexes to normal AML1 target genes. Therefore, the identification of individual genes or biological pathways that are specifically disrupted in the presence of AML1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to the possibility for improved treatment for these patients.We identified RASSF2 as a gene that is specifically downregulated in (2-4 fold) in total bone marrow of t(8;21) patients compared to non-t(8;21) FAB-subtype M2 AML patients by analyzing publicly available gene expression datasets. Similarly, using a mouse model of t(8;21) AML we found Rassf2 mRNA levels to be nearly 30-fold lower in t(8;21) leukemia cells compared to wild-type Lin-Sca-cKit+ (LK) myeloid progenitors. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 mRNA levels are significantly downregulated (8-10-fold) in both Kasumi-1 and SKNO-1 t(8;21) cell lines as compared to a similar non-t(8;21) HL-60 cell line and to primary human CD34+ control cells. In addition, expression of AML1-ETO in HL-60 or CD34+ cells results in a decrease in RASSF2 mRNA expression, which further suggests that RASSF2 is a target for regulation by AML1-ETO. Assessment of published ChIP-seq data shows that AML1-ETO binds the RASSF2 gene locus at two distinct regions in both primary t(8;21) AML patient samples and in the Kasumi-1 and SKNO-1 cell lines. These regions are similarly bound by several important hematopoietic transcription factors in primary human CD34+ cells, including AML1, ERG, FLI1, and TCF7L2, implicating these two regions as important for the regulation of RASSF2 expression during blood cell differentiation. Overexpression of RASSF2 in human leukemia cell lines using an MSCV-IRES-GFP (MIG) construct revealed that RASSF2 has a strong negative effect on leukemia cell proliferation and viability. The overall percentage of GFP-positive cells in MIG-RASSF2 transduced cells markedly decreased compared to MIG-control transduced cells over a period of 14 days. This effect was primarily due to significantly increased apoptosis in the RASSF2 expressing cell populations. Similarly, we found that expression of RASSF2 significantly inhibits the long-term self-renewal capability of hematopoietic cells transduced with AML1-ETO in a serial replating/colony formation assay. AML1-ETO transduced hematopoietic cells were normally capable of serial replating for more than 6 weeks. However, AML1-ETO transduced cells co-expressing RASSF2 consistently had reduced colony number and lost their ability to replate after 3-4 weeks. This was due to a dramatically increased rate of apoptosis in RASSF2 expressing cells.RASSF2 is reported to be a tumor suppressor that is frequently downregulated at the transcriptional level by hypermethylation in primary tumor samples, but not healthy controls. Here we have identified RASSF2 as a target for repression, and demonstrated its tumor suppressive function in t(8;21) leukemia cells. Further insights into the molecular mechanisms of RASSF2 function in AML will continue to be explored. DisclosuresNo relevant conflicts of interest to declare.

Clinical Impact of Additional Cytogenetic Aberrations, cKIT- and RAS Mutations and Other Factors in Pediatric t(8;21)-AML

IntroductionWhilst pediatric t(8;21)-positive AML has an overall survival rate of up to 80-90%, the relapse rate at 30-40% remains unacceptably high. Factors that reliably discriminate between patients with t(8;21) who do or do not relapse have been poorly described. The prognostic impact of additional cytogenetic aberrations and of cKIT or RAS mutations in this subgroup remains unclear. In this large retrospective cohort study, supported by the International-BFM study group, we aimed firstly to determine outcome in relation to these aberrations and, secondly to identify any treatments associated with outcome.MethodsData on diagnostics, treatment, response, relapse or other events and follow-up were collected from 19 childhood cancer study groups, who provided data from 916 pediatric AML-patients positive for t(8;21) and/or AML1-ETO fusion gene. Karyotypes were reviewed and classified (CH&BB). Aberrations occurring in at least 10 patients were considered for analyses and involved loss of sex chromosome, deletions of chromosome 9q (del9(q)), abnormalities of chromosome 7q, gain of chromosome 4 (+4) and gain of chromosome 8. A total of 228 samples could be evaluated for cKIT and/or RAS mutations. Significant characteristics in explorative analysis were used in 2 multivariate Cox proportional-hazard regression models for assessing effects of induction and overall treatment elements. Induction was defined as first two courses of chemotherapy. Endpoints were probabilities of achieving complete remission (pCR), of event free survival (pEFS), and of overall survival (pOS) and cumulative incidence of relapse (CIR). Only results of multivariate analyses are shown.ResultsIn final analyses 838 patients with a full karyotype were included. CR was achieved in 92% of the patients. Five-year pOS was 74% (SE 1.5%), five-year pEFS 58% (SE 1.7%) and five-year CIR 26% (SE 1.5%). WBC count >20 x109/L was associated with poorer outcome for all endpoints (p<0.01). CNS involvement at presentation led to higher CIR (p<0.01). Twenty five percent of the patients had cKIT mutation(s). No differences in clinical or leukemic characteristics between mutated and wild-type patients were identified. Seven out of 10 patients with +4 had cKIT mutation(s) (p<0.01). Patients with RAS mutations (13%) had higher WBC counts (p=0.03). No clinical differences between N-RAS (n=17) and K-RAS (n=5) mutated patients were found. No associations between other additional aberrations and cKIT- or RAS mutations were identified. Patients with del(9)(q) (n=104) had lower pCR (p=0.01), without influencing pEFS or pOS. Additional +4 (n=21) was strongly associated with higher CIR (p<0.01) and poorer survival rates (5ypEFS 18% versus 59%, plogrank<0.01 and 5ypOS 33% versus 75%, plogrank<0.01). Presence of other cytogenetic aberrations including complex karyotype or cKIT or RAS mutations did not affect outcome.Anthracyclines >150mg/m2 used in first induction course was associated with higher pCR (p=0.04) and higher pOS (p=0.03). Etoposide >500mg/m2 used in first course was associated with higher pEFS (p=0.01) and pOS (p=0.02). The use of HD-AraC 3g/m2 during induction was associated with higher pCR (p=0.02). When including consolidation therapy, cumulative dosages of cytarabine >30g/m2 and etoposide >1500mg/m2 were associated with lower CIR (p=0.02 and p<0.01 respectively).ConclusionThis study identifies novel negative prognostic factors in pediatric t(8;21)-AML patients - del(9q) associated with an inferior pCR, and +4 significantly associated with an increased relapse rate and inferior survival. This challenges the favorable classification of patients with t(8;21) and +4. The presence of del(9q) may indicate the need for intensified induction therapy in view of the lower pCR. Although cKIT or RAS mutations did not influence outcome in conventionally treated t(8;21)-patients in this study, mutated patients may benefit further from treatment with tyrosine kinase inhibitors.This study suggests benefit from protocols containing high-dose anthracyclines, etoposide and HD-AraC 3g/m2 during induction, and the use of cumulative dosages of cytarabine >30g/m2 and of etoposide >1500mg/m2 in pediatric t(8;21)-positive AML. Optimal dosages and combinations of these agents should be established in prospective randomized trials. DisclosuresNo relevant conflicts of interest to declare.

Could Anthracycline be Replaced in Chemotherapy for Acute Myeloid Leukemia? Randomized Clinical Trial of Homoharringtonine As Frontline Agent to Treat Pediatric AML

Method: From Jan. 1st , 2009 to Dec. 31st, 2013, 153 patients was diagnosed to be acute myeloid leukemia (non acute promyelocytic leukemia) (AML). 152 of them were willing to be recruited in the randomized clinical trial. After informed consent, randomly divided group A and group B were treated with regimens A and B accordingly. In regimen A, total 240mg/m2 daunorubicin and 60 mg/m2 mitoxantrone were given in total 6 cycles of intensive chemotherapy along with cytarabine and/or etopside; while in regimen B, the anthracycline was substituted for homoharringtonine except the first cycle, ie, only 120 mg/m2 daunorubicin was used in regimen B. All the patients recruited were registered to Pediatric Oncology Network Database (POND) provided by St. Jude Children’s Research Hospital once the diagnose was made and the consent form was signed. Regular follow-up was done every 3 months for patients off treatment. The end point of this follow-up was 2014/6/30.Besides the regular cytogenetic study of all cases, c-kit mutations in exon 8 and exon 17 were screened by direct sequencing.Results: There were 82 and 70 cases de novo AML in group A and group B respectively. The distribution of FAB subtypes and the major cytogenetic abnormalities was similar (p>0.1) between the groups. For the first course in both regiment were the same, the similarity of the first course remission rate (p=0.2), hematological changes and the febrile neutropenia occurrence in this cycle of chemotherapy may reflect the intrinsic accordance of two groups. Analyzing the survival function of the protocol, the 3-year Events free survival (EFS) was 59.3±5.1%, 3 year overall survival rate (OS) was 73 ± 4.8%, and 25.6 ± 5% patients finally relapsed. Comparing the group A and the group B, EFS, OS and relapse rate were similar: 62.6 ± 6.8% Vs 58 ± 7.5%, 76.1 ± 6.3% Vs 70.1 ± 7.2%, and 27.7 ± 7% Vs 28.3 ± 7.4%, P values were more than 0.1. Interestingly, except the first cycle induction therapy, all the regiment B course had less hematological toxicity and less incidence and severity of febrile neutropenia. Group A Vs group B, the average duration of neutrophil count <0.5 x 109/L in each course was 11.3 ± 0.8 Vs 8.5 ± 0.7 days, p=0.0004; duration of platelet count <50 x 109/L was 12.4 ± 1.5 Vs 7.8 ± 0.9 days, p=0.0003; while the incidence of febrile neutropenia was 48% Vs 23% p<0.0001 and the severe infection was occurred in 2.9% group A patients, compared to only in 0.5% group B patients, p=0.042.During prognosis analysis, we surprisingly found that patients with AML1-ETO fusion gene, which was well accepted as a favorable prognosis predictor, often have worse outcome: the 3-year EFS was only 43.4 ± 8.6%, much lower than which of the Non-AML1-ETO patient (68.3±6.1%, p=0.024). It might be partially due to the higher frequency of c-Kit mutation: in these 43 AML1-ETO positive patients, 18 (41.8%)were found to have mutation in c-Kit at exon 8 and exon 17. These mutants usually accompanied with poorer survival especially those with mutation at exon 8 and D816. The 3-year EFS of patients with these two kinds of mutation were 13 ± 12.1% compared to the other AML1-ETO+ patients’ 57.2 ± 10.3%. But still, the outcome of these AML1-ETO+ patients without bad c-kit mutation were worse than patients with normal karyotype, which had 3-EFS 76.4 ± 10.7%. In this study, the survival feature of patients with AML1-ETO was regimenting independent.Conclusion: 1) The efficacy of homoharringtonine contacting regiment was similar to the standard anthracycline contacting regiment. The former usually had less toxicity. It may helpful for improving the patient’s quality of life, reduce the economic burden of patients’ family without compromising the survival. 2) For Chinese children, AML1-ETO fusion gene is not a good prognosis predictor, at least in part, due to more conmen c-Kit mutation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PPAR-γ Agonists Induce Growth Arrest of Kit/AML1-ETO Leukemia Cells Acting on Diverse Pathways

Translocation 8;21, t(8;21), with an incidence of up to 12% is among the most common genetic aberrations found in Acute Myeloid leukemias (AMLs). t(8;21) rearranges the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generating an AML1-ETO fusion protein. It acts as dominant-negative repressor of genes involved in cell cycle control and myeloid differentiation. Despite its critical role in tumor initiation, AML1-ETO is not sufficient, per se, to cause an overt leukemia. It has to cooperate with other genetic alterations, such as those involving receptor tyrosine kinases. KIT tyrosine kinase gain-of-function mutations have been detected in about 50% of t(8;21) AMLs.Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) agonists are known to inhibit proliferation and induce apoptosis in cancer cells, including those from hematologic malignancies. Despite several promising pre-clinical studies, results of clinical trials are not conclusive so far.We investigated the effects and mechanisms of action of some PPAR-γ agonists in an AML1-ETO/Kit activated leukemia cell line: Kasumi-1. Troglitazone (TGZ) and rosiglitazone (RGZ) reduced proliferation rate and clonogenic potential of Kasumi-1, inducing G1 arrest and apoptosis in a dose and time dependent manner. Activated Kit induces proliferation mostly through the Src kinases/Ras/MAPK pathway. Erk1/2 is constitutively phosphorylated in Kasumi-1 cells and exposure to troglitazone (50μM) significantly reduced its phosphorylated state even if not as much as imatinib. The Kit signaling cascades include activation of PI3-kinase/AKT pathway, but surprisingly TGZ treatment did not affect AKT phosphorylation.It has been shown that PPAR agonists 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) and 15-Deoxy-Δ12,14-ProstaglandinJ2 (15d-PGJ2) are able to suppress TGFβ-induced phosphorylation of AktS473, leading an anti-fibrotic activity in the lung. CDDO and 15d-PGJ2 inhibition effect have been demonstrated direct, PPAR-γindependent and mostly related to electrophilic carbons present in their structures. In the same cellular context rosiglitazone was found relatively poorly effective. We compared, in Kasumi-1 cells, activity of 15d-PGJ2 and CAY10410, two PPAR agonists with similar structure but while 15d-PGJ2 has an electrophilic center CAY10410 does not. CAY10410 is a strong PPAR agonist but was unable to induce any reduction in Kasumi-1 cells proliferation rate or induce apoptosis whereas 15d-PGJ2 strongly inhibited Kasumi-1 growth and induced apoptosis even more efficiently than troglitazone. Molecular characterization of such functional effect is of extreme interest and is ongoing.TGZ and RGZ induced up-regulation of p27Kip1 and p21cip1. We found that it occurs by reduced proteosomal degradation and accumulation of p27kip1 due to up-regulation of Connexin 43 (Cx43) and consequent downregulation of Skp2. AML1-ETO has been reported as Cx43 transcriptional inducer. Upregulation of p21Waf1 seems to be, in preliminary experiments, mostly transcriptional and AML1-ETO direct induction of p21Waf1has been described as well. Interestingly TGZ seems to restore AML1-ETO functions counteracted upon Kit constitutive activation.Some, if not all, the effects might be receptor independent since they were just minimally lessened by a pre-treatment with high dose of GW9662, an irreversible PPAR-γ antagonist that covalently binds to the active site of PPAR-γ.The overall prognosis of AML patients remains severe and AML incidence is expected to increase with the aging population, forcing the need for less toxic regimens in patients with co-morbid conditions precluding intensive chemotherapy. PPAR agonists are promising candidates as specific anticancer drugs within low toxicity multidrug regimens. However, the effectiveness of PPAR ligands seems to vary as various compounds, targeting distinct leukemic cells, behavior differently, because different are the leading molecular mechanism evoked. Their precise clarification will define the real extent of PPAR agonists’ therapeutic potential. DisclosuresNo relevant conflicts of interest to declare.

CBL restricts myeloid proliferation of human AML1-ETO preleukemia cells

CBL is a multifunctional protein engaged in the regulation of intracellular signaling pathways. Recent studies have found CBL mutations and deletions in a variety of myeloid diseases. In acute myeloid leukemia (AML), CBL mutations are associated with core binding factor (CBF) leukemia, a subset of AML characterized by the presence of AML1-ETO or CBFB-MYH11 fusion genes. However, the potential cooperation between mutant CBL and CBF-fusion proteins has not been demonstrated experimentally.