The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia.
ABSTRACT: Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.
Project description:Purpose: Identify new targets in EVI1-Positive Acute Myeloid Leukemia Methods: Treatment with cyclocreatine of EVI1-driven AML cell lines TF-1, UT-7 and UCSD-AML1. Cyclocreatine was purchased from Sigma-Aldrich (Sigma). TF-1, UT-7 and UCSD-AML1 cells were treated in quadruplicate with either vehicle or 3 mM cyclocreatine for 24 hours. Total RNA was extracted and profiled by RNA sequencing (HiSeq, Illumina) at BioMicroCenter from Massachusetts Institute of Technology (Cambridge, MA, USA). Results: Alteration of arginine-creatine metabolism by the small-molecule cyclocreatine selectively decreased the viability, promoted cell cycle arrest and apoptosis of EVI1-positive AML cells. Conclusions: Targeting CKMT1 is a promising therapeutic strategy for the EVI1-driven AML subtype that is highly resistant to current treatment regimens. Overall design: mRNA profiles for the EVI1-driven AML cell lines TF-1, UT-7 and UCSD-AML1 treated with either 3mM cyclocreatine for 24 hours or with vehicle were generated in quadruplicate by RNA sequencing (HiSeq, Illumina) at the BioMicroCenter from Massachusetts Institute of Technology (Cambridge, MA, USA).
Project description:Chromosomal rearrangements between 3q21 and 3q26 elicit high-risk acute myeloid leukemia (AML), which is often associated with elevated platelet and megakaryocyte (Mk) numbers. The 3q rearrangements reposition a GATA2 enhancer near the EVI1 (or MECOM) locus, which results in both EVI1 overexpression and GATA2 haploinsufficiency. However, the mechanisms explaining how the misexpression of these 2 genes individually contribute to leukemogenesis are unknown. To clarify the characteristics of differentiation defects caused by EVI1 and GATA2 misexpression and to identify the cellular origin of leukemic cells, we generated a system to monitor both inv(3) allele-driven EVI1 and Gata2 expression in 3q-rearranged AML model mice. A cell population in which both EVI1 and Gata2 were highly induced appeared in the bone marrows before the onset of frank leukemia. This population had acquired serial colony-forming potential. Because hematopoietic stem/progenitor cells (HSPCs) and Mks were enriched in this peculiar population, we analyzed the independent EVI1 and GATA2 contributions to HSPC and Mk. We found that inv(3)-driven EVI1 promotes accumulation of Mk-biased and myeloid-biased progenitors, Mks, and platelets, and that Gata2 heterozygous deletion enhanced Mk-lineage skewing of EVI1-expressing progenitors. Notably, inv(3)-directed EVI1 expression and Gata2 haploinsufficient expression cooperatively provoke a leukemia characterized by abundant Mks and platelets. These hematological features of the mouse model phenocopy those observed in human 3q AML. On the basis of these results, we conclude that inv(3)-driven EVI1 expression in HSPCs and Mks collaborates with Gata2 haploinsufficiency to provoke Mk-lineage skewing and leukemogenesis with excessive platelets, thus mimicking an important feature of human AML.
Project description:Aberrantly high expression of EVI1 in acute myeloid leukaemia (AML) is associated with poor prognosis. For targeted treatment of EVI1 overexpressing AML a more detailed understanding of aspects of spatiotemporal interaction dynamics of the EVI1 protein is important. EVI1 overexpressing SB1690CB AML cells were used for quantification and protein interaction studies of EVI1 and ?EVI1. Cells were cell cycle-synchronised by mimosine and nocodazole treatment and expression of EVI1 and related proteins assessed by western blot, immunoprecipitation and immunofluorescence. EVI1 protein levels oscillate through the cell cycle, and EVI1 is degraded partly by the proteasome complex. Both EVI1 and ?EVI1 interact with the co-repressor CtBP1 but dissociate from CtBP1 complexes during mitosis. Furthermore, a large fraction of EVI1, but not ?EVI1 or CtBP1, resides in the nuclear matrix. In conclusion, EVI1- protein levels and EVI1-CtBP1 interaction dynamics vary though the cell cycle and differ between EVI1 and ?EVI1. These data ad to the functional characterisation of the EVI1 protein in AML and will be important for the development of targeted therapeutic approaches for EVI1-driven AML.
Project description:EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-?-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15(INK) (4B) by transforming growth factor (TGF)-? and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-?-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-?-mediated growth inhibition of HCC cells.
Project description:MicroRNA-22 (miR-22) is emerging as a critical regulator in organ development and various cancers. However, its role in normal hematopoiesis and leukaemogenesis remains unclear. Here, we detected its increased expression during monocyte/macrophage differentiation of HL-60, THP1 cells and CD34+ hematopoietic stem/progenitor cells, and confirmed that PU.1, a key transcriptional factor for monocyte/macrophage differentiation, is responsible for transcriptional activation of miR-22 during the differentiation. By gain- and loss-of-function experiments, we demonstrated that miR-22 promoted monocyte/macrophage differentiation, and MECOM (EVI1) mRNA is a direct target of miR-22 and MECOM (EVI1) functions as a negative regulator in the differentiation. The miR-22-mediated MECOM degradation increased c-Jun but decreased GATA2 expression, which results in increased interaction between c-Jun and PU.1 via increasing c-Jun levels and relief of MECOM- and GATA2-mediated interference in the interaction, and thus promoting monocyte/macrophage differentiation. We also observed significantly down-regulation of PU.1 and miR-22 as well as significantly up-regulation of MECOM in acute myeloid leukemia (AML) patients. Reintroduction of miR-22 relieved the differentiation blockage and inhibited the growth of bone marrow blasts of AML patients. Our results revealed new function and mechanism of miR-22 in normal hematopoiesis and AML development and demonstrated its potential value in AML diagnosis and therapy.
Project description:Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome, characterized by thrombocytopenia and congenital fusion of the radius and ulna. A heterozygous HOXA11 mutation has been identified in two unrelated families as a cause of RUSAT. However, HOXA11 mutations are absent in a number of individuals with RUSAT, which suggests that other genetic loci contribute to RUSAT. In the current study, we performed whole exome sequencing in an individual with RUSAT and her healthy parents and identified a de novo missense mutation in MECOM, encoding EVI1, in the individual with RUSAT. Subsequent analysis of MECOM in two other individuals with RUSAT revealed two additional missense mutations. These three mutations were clustered within the 8(th) zinc finger motif of the C-terminal zinc finger domain of EVI1. Chromatin immunoprecipitation and qPCR assays of the regions harboring the ETS-like motif that is known as an EVI1 binding site showed a reduction in immunoprecipitated DNA for two EVI1 mutants compared with wild-type EVI1. Furthermore, reporter assays showed that MECOM mutations led to alterations in both AP-1- and TGF-?-mediated transcriptional responses. These functional assays suggest that transcriptional dysregulation by mutant EVI1 could be associated with the development of RUSAT. We report missense mutations in MECOM resulting in a Mendelian disorder that provide compelling evidence for the critical role of EVI1 in normal hematopoiesis and in the development of forelimbs and fingers in humans.
Project description:DNA methylation patterns are frequently dysregulated in cancer, although little is known of the mechanisms through which specific gene sets become aberrantly methylated. The ecotropic viral integration site 1 (EVI1) locus encodes a DNA binding zinc-finger transcription factor that is aberrantly expressed in a subset of acute myeloid leukemia (AML) patients with poor outcome. We find that the promoter DNA methylation signature of EVI1 AML blast cells differs from those of normal CD34(+) bone marrow cells and other AMLs. This signature contained 294 differentially methylated genes, of which 238 (81%) were coordinately hypermethylated. An unbiased motif analysis revealed an overrepresentation of EVI1 binding sites among these aberrantly hypermethylated loci. EVI1 was capable of binding to these promoters in 2 different EVI1-expressing cell lines, whereas no binding was observed in an EVI1-negative cell line. Furthermore, EVI1 was observed to interact with DNA methyl transferases 3A and 3B. Among the EVI1 AML cases, 2 subgroups were recognized, of which 1 contained AMLs with many more methylated genes, which was associated with significantly higher levels of EVI1 than in the cases of the other subgroup. Our data point to a role for EVI1 in directing aberrant promoter DNA methylation patterning in EVI1 AMLs.
Project description:The ecotropic virus integration site 1 (EVI1) transcription factor is associated with human myeloid malignancy of poor prognosis and is overexpressed in 8-10% of adult AML and strikingly up to 27% of pediatric MLL-rearranged leukemias. For the first time, we report comprehensive genomewide EVI1 binding and whole transcriptome gene deregulation in leukemic cells using a combination of ChIP-Seq and RNA-Seq expression profiling. We found disruption of terminal myeloid differentiation and cell cycle regulation to be prominent in EVI-induced leukemogenesis. Specifically, we identified EVI1 directly binds to and downregulates the master myeloid differentiation gene Cebpe and several of its downstream gene targets critical for terminal myeloid differentiation. We also found EVI1 binds to and downregulates Serpinb2 as well as numerous genes involved in the Jak-Stat signaling pathway. Finally, we identified decreased expression of several ATP-dependent P2X purinoreceptors genes involved in apoptosis mechanisms. These findings provide a foundation for future study of potential therapeutic gene targets for EVI1-induced leukemia.
Project description:BACKGROUND:The transcription factor Ecotropic Virus Integration site 1 (EVI1) regulates cellular proliferation, differentiation, and apoptosis, and its overexpression contributes to an aggressive course of disease in myeloid leukemias and other malignancies. Notwithstanding, knowledge about the target genes mediating its biological and pathological functions remains limited. We therefore aimed to identify and characterize novel EVI1 target genes in human myeloid cells. METHODS:U937T_EVI1, a human myeloid cell line expressing EVI1 in a tetracycline regulable manner, was subjected to gene expression profiling. qRT-PCR was used to confirm the regulation of membrane-spanning-4-domains subfamily-A member-3 (MS4A3) by EVI1. Reporter constructs containing various parts of the MS4A3 upstream region were employed in luciferase assays, and binding of EVI1 to the MS4A3 promoter was investigated by chromatin immunoprecipitation. U937 derivative cell lines experimentally expressing EVI1 and/or MS4A3 were generated by retroviral transduction, and tested for their tumorigenicity by subcutaneous injection into severe combined immunodeficient mice. RESULTS:Gene expression microarray analysis identified 27 unique genes that were up-regulated, and 29 unique genes that were down-regulated, in response to EVI1 induction in the human myeloid cell line U937T. The most strongly repressed gene was MS4A3, and its down-regulation by EVI1 was confirmed by qRT-PCR in additional, independent experimental model systems. MS4A3 mRNA levels were also negatively correlated with those of EVI1 in several published AML data sets. Reporter gene assays and chromatin immunoprecipitation showed that EVI1 regulated MS4A3 via direct binding to a promoter proximal region. Experimental re-expression of MS4A3 in an EVI1 overexpressing cell line counteracted the tumor promoting effect of EVI1 in a murine xenograft model by increasing the rate of apoptosis. CONCLUSIONS:Our data reveal MS4A3 as a novel direct target of EVI1 in human myeloid cells, and show that its repression plays a role in EVI1 mediated tumor aggressiveness.
Project description:Amplification of 3q26.2, found in many cancer lineages, is a frequent and early event in ovarian cancer. We previously defined the most frequent region of copy number increase at 3q26.2 to EVI1 (ecotropic viral integration site-1) and MDS1 (myelodysplastic syndrome 1) (aka MECOM), an observation recently confirmed by the cancer genome atlas (TCGA). MECOM is increased at the DNA, RNA, and protein level and likely contributes to patient outcome. Herein, we report that EVI1 is aberrantly spliced, generating multiple variants including a Del(190-515) variant (equivalent to previously reported) expressed in >90% of advanced stage serous epithelial ovarian cancers. Although EVI1(Del190-515) lacks ?70% of exon 7, it binds CtBP1 as well as SMAD3, important mediators of TGF? signaling, similar to wild type EVI1. This contrasts with EVI1 1-268 which failed to interact with CtBP1. Interestingly, the EVI1(Del190-515) splice variant preferentially localizes to PML nuclear bodies compared to wild type and EVI1(Del427-515). While wild type EVI1 efficiently repressed TGF?-mediated AP-1 (activator protein-1) and plasminogen activator inhibitor-1 (PAI-1) promoters, EVI1(Del190-515) elicited a slight increase in both promoter activities. Expression of EVI1 and EVI1(Del427-515) (but not EVI1(Del190-515)) in OVCAR8 ovarian cancer cells increased cyclin E1 LMW expression and cell cycle progression. Furthermore, knockdown of specific EVI1 splice variants (both MDS1/EVI1 and EVI1(Del190-515)) markedly increased claudin-1 mRNA and protein expression in HEY ovarian and MDA-MB-231 breast cancer cells. Changes in claudin-1 were associated with alterations in specific epithelial-mesenchymal transition markers concurrent with reduced migratory potential. Collectively, EVI1 is frequently aberrantly spliced in ovarian cancer with specific forms eliciting altered functions which could potentially contribute to ovarian cancer pathophysiology.