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Myeloid ELF1-like factor (MEF), also known as ELF4, is a member of the ETS family of transcription factors which is expressed in hematopoietic cells. MEF-deficient mice have defects in natural killer cell and natural killer T cell development, suggesting a role for MEF in regulating innate immunity. MEF also functions in myeloid cells, where it can transactivate target genes. To identify MEF target genes in a "myeloid" environment, we created an inducible expression system and used oligonucleotide microarrays to examine the transcript profile of HEL cells after induction of MEF expression. Sixteen genes were reproducibly turned on or off more than 2-fold, 8 h after induction of MEF expression, and we examined one of the genes, interleukin-8 (IL-8), in greater detail. IL-8 is a CXC chemokine involved in neutrophil chemoattraction, angiogenesis, and stem cell mobilization. It is expressed by several tumor types, and its expression is regulated primarily transcriptionally. The IL-8 promoter contains three ETS binding sites, and we identified the specific site that binds MEF and is required for MEF responsiveness. MEF, but not the closely related ETS factors PEA3, ETS1, ETS2, ELF1, or PU.1, strongly activates the IL-8 promoter. MEF overexpression is sufficient to induce IL-8 protein expression, and reduction in MEF expression (using RNA interference) results in decreased IL-8 levels. These data demonstrates that MEF is an important regulator of IL-8 expression.

Prospective randomized studies have determined that high dose therapy and autologous stem cell transplantation (ASCT) is the standard treatment for patients with chemosensitive relapsed Hodgkin's disease (HD); however, the role of this approach in patients with primary refractory disease has been controversial. This report is an integrated analysis of 75 consecutive patients with biopsy-confirmed primary refractory HD, who were treated with high dose chemoradiotherapy (HDT) and ASCT at the Memorial Sloan Kettering Cancer Center. The patients underwent conventional dose cytoreductive chemotherapy followed by HDT and ASCT. At a median follow-up of 10 years for surviving patients, the event-free survival (EFS), progression-free survival (PFS) and overall survival (OS) rates were 45%, 49% and 48% respectively. Only chemosensitivity to standard-dose second-line chemotherapy (SDSC) predicted for a better survival, thus responding patients had an EFS, PFS and OS of 60%, 62% and 66%, respectively, versus 19%, 23% and 17% for patients who had a poor response to SDSC (P < 0.001). While patients with chemosensitive disease have an excellent outcome with HDT and ASCT, novel approaches are needed to cure HD patients who fail front-line and second-line chemotherapy.

Hematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

The role of autologous stem cell transplantation (AuSCT) in older multiple myeloma patients is unclear. Using data from the Autologous Blood and Marrow Transplant Registry, we compared the outcome of 110 patients/=the age of 60 (median 63; range 60-73) years, undergoing AuSCT with that of 382 patients60 (median 52; range 30-59) years. The two groups were similar except that older patients had a higher beta(2)-microglobulin level at diagnosis (P=0.016) and fewer had lytic lesions (P=0.007). Day 100 mortality was 6% (95% confidence interval 4-9) and 1-year treatment-related mortality (TRM) was 9% (6-13) in patients60 years, compared with 5% (2-10) and 8% (4-14), respectively, in patients/=60 years. The relapse rate, progression-free survival (PFS) and overall survival (OS) in the two groups were also similar. Multivariate analysis of all patients identified only an interval from diagnosis to AuSCT12 months and the use of two prior chemotherapy regimens within 6 months of AuSCT as adverse prognostic factors. Our results indicate that AuSCT can be safely performed in selected older patients: the best results were observed in patients undergoing AuSCT relatively early in their disease course.

MEF (myeloid Elf-1 like factor) is a member of the ETS family of transcription factors (TF) with transcriptional activating properties. ETS proteins have been implicated in widely divergent physiological and pathological processes (such as development and oncogenesis). MEF is expressed in non-hematopoietic and hematopoietic (lymphoid and myeloid) tissues, and after generating MEF-deficient mice by homologous recombination, we have studied its role in lymphopoiesis (Immunity 17 (2002), 437). MEF plays a critical role in NK and NK-T cell development and the constitutive expression of perforin by NK cells. MEF interacts with other TFs such as AML1 (Runx1) and with the cyclin A/cdk2 kinase complex. In this review, we discuss the biology of MEF in the context of the other members of this family of transcriptional regulators.

Summary. Malignant plasma cells generally grow within the bone marrow microenvironment; however, they can also grow at extramedullary sites. To identify the tumour-specific alterations required for extramedullary growth, we analysed the expression profiles of a series of plasma cell neoplasms including primary multiple myeloma (MM), plasma cell leukaemia (PCL) and extramedullary plasmacytoma (EPC). Hierarchical clustering analysis segregated the EPCs from the remaining samples, and revealed an expression pattern associated with angiogenesis in the EPCs, involving higher expression of the genes TIE2, NOTCH3, CD31 and endoglin. Direct comparison of EPC samples with the MM samples identified 156 genes significantly upregulated and 85 genes significantly downregulated (P

Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Valpha24+/Vbeta11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon-gamma-producing NKT cells in response to the NKT ligand, alpha-galactosyl ceramide, although influenza-virus-specific effector T-cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.

We report on the X-ray structure of three 100-amino acid mbt repeats in h-l(3)mbt, a polycomb group protein involved in transcriptional repression, whose gene is located in a region of chromosome 20 associated with hematopoietic malignancies. Interdigitation between the extended arms and cores of the mbt repeats results in a three-leaved propeller-like architecture, containing a central cavity. We have identified one ligand binding pocket per mbt repeat, which accommodates either the morphilino ring of MES or the proline ring of the C-terminal peptide segment, within a cavity lined by aromatic amino acids. Strikingly, phenotypic alterations resulting from point mutations or deletions in the mbt repeats of the related Drosophila SCM protein are clustered in and around the ligand binding pocket.

ETS proteins (such as PU.1, Fli-1 and ETS-1) have been shown to play important roles in normal and abnormal hematopoiesis. We examined the expression of the ELF subfamily of ETS genes (ELF-1, MEF and NERF) in acute myeloid leukemia (AML) cells using Northern blot analysis. ELF-1 and MEF were expressed in all samples, whereas NERF was not. The relative expression (RE) of MEF, but not ELF-1, was significantly lower (P

Despite advances in the management of aggressive non-Hodgkin's lymphoma, the treatment of relapsed and primary refractory disease remains a major challenge. High-dose chemotherapy or radio-chemotherapy followed by autologous or allogeneic stem cell transplantation (SCT) is a potentially curative treatment approach; however, the applicability of this approach is restricted to patients responding to second-line chemotherapy. Thus, second-line therapy must be both efficacious and without stem cell or organ toxicity that would compromise the ability to proceed to SCT. The ifosfamide, carboplatin and etoposide (ICE) regimen was developed to address these challenges. In a series of prospective clinical trials, 222 patients were treated with the ICE regimen. with an overall response rate of 72%. The mobilization of stem cells with this regimen was excellent,with 86% of patients mobilizing at least 2.0 x 10(6) CD34+ cells/kg. The incidence of treatment-related toxicity precluding SCT after ICE is very low. Herein, we report the clinical results of this treatment program for 222 patients with 5-year median follow-up for surviving patients. Rituximab was subsequently added to the ICE regimen for patients with diffuse large B-cell lymphoma (DLBCL) to improve upon these favorable results. This resulted in an increased complete remission rate. Additional follow-up is necessary to determine if this improvement in the complete remission rate will confer an increase in the overall survival following SCT.

The CCAAT/enhancer binding protein alpha (C/EBPalpha) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPalpha(-/-) mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPalpha in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPalpha gene and down-regulation of C/EBPalpha by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPalpha in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34(+) cells with a retroviral construct that expresses the C/EBPalpha cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPalpha function in primary human CD34(+) cells, by the addition of beta-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPalpha target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor of differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.

Erythroid leukemia (ERL or AML-M6) is an uncommon subtype of acute myeloid leukemia, the clinical, morphological, and genetic behavior of which needs further characterization. We analyzed a homogeneous group of 23 de novo AML-M6 patients whose bone marrow cells showed complex karyotypes. We also analyzed eight leukemia cell lines with erythroid phenotype, performing detailed molecular cytogenetic analyses, including spectral karyotyping (SKY) in all samples. The main features are: (1) A majority of patients (56%) had hypodiploidy. Loss of genetic material was the most common genetic change, especially monosomies of chromosome 7 or 18, and deletions of chromosome arm 5q. Taken together, 87% of the cases displayed aberrations involving chromosome 5 or 8. (2) We describe a novel, cryptic, and recurrent translocation, t(11;19)(p11.2;q13.1). Another translocation, t(12;21)(p11.2;q11.2), was found to be recurrent in a patient with ERL and in the K562 cell line. (3) MLL gene rearrangements were detected in 20% of cases (three translocations and three amplifications) and, overall, we defined 52 rearrangements (excluding deletions) with a mean of 2.3 translocations per patient. (4) Of the structural aberrations, 21% involved chromosomes 11 and 19. Most of the rearrangements were unbalanced; only 13 reciprocal translocations were observed. The general picture of chromosomal aberrations in cell lines did not reflect what occurred in patient samples. However, both primary samples and cell lines shared three common breakpoints at 19q13.1, 20q11.2, and 21q11.2. This is the first molecular cytogenetic description of the karyotype abnormalities present in patients with ERL. It should assist in the identification of genes involved in erythroleukemogenesis. © 2003 Wiley-Liss, Inc.

We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF−/− mice have a profound reduction in the number of NK-T and NK cells. Purified MEF−/− NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNγ. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.

Chromatin modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs) have emerged as important targets in cancer. PRMT4, also known as CARM1, is overexpressed in a number of cancers, including breast, prostate, pancreatic, and lung cancer. Our lab reported the overexpression of PRMT4 in the context of acute myeloid leukemia (AML), showing that more than 70% of cytogenetically normal AML patients have up-regulation of PRMT4. Here, we investigated the role of PRMT4 in normal hematopoiesis and leukemia development. In order to study the role of PRMT4 in normal hematopoiesis, Prmt4-floxed mice were crossed with Vav1-cre mice purchased from the Jackson Laboratory. Inducible Prmt4 conditional KO mice were generated by crossing Prmt4-floxed mice with Mx1-Cre mice and Prmt4 gene excision was induced by poly(I:C). Using this hematopoietic specific knockout system, we show that loss of PRMT4 has little effect on normal hematopoiesis, but promotes the differentiation of hematopoietic stem and progenitor cells. Next we evaluated the role of PRMT4 in leukemia development using leukemia transplantation models driven by fusion oncoproteins. Strikingly, the knockout of PRMT4 completely abrogates leukemia initiation in fetal liver transplantation models driven by the AML1-ETO or MLL-AF9 fusion proteins. We further characterized the mechanism for the leukemia-specific dependence on PRMT4 using leukemia cell lines and found that knockdown of PRMT4 impairs cell cycle progression, decreases proliferation, and induces rapid apoptosis. To examine PRMT4 dependent changes in gene expression in a leukemia system, we used lentiviral vectors that express RFP and shRNAs directed against PRMT4. We knocked down PRMT4 in four leukemia cell lines or normal human cord-blood derived CD34+ cells. Gene set enrichment analysis showed that all four leukemia cell lines with knockdown of PRMT4 significantly down-regulated E2F target genes compared to the scrambled control. Chromatin immunoprecipitation analysis (ChIP) confirmed the presence of PRMT4 and H3R17 dimethylation at the promoter regions of E2F1 targets. The PRMT4 conditional knockout mice and PRMT4 knockdown experiments both suggest that the loss of PRMT4 protein has a selective effect on leukemia cells compared to normal hematopoietic stem and progenitor cells. Collectively, this work suggests that targeting PRMT4 through chemical inhibition may be an effective therapeutic strategy for AML and other cancers with up-regulation of PRMT4. Citation Format: Sarah M. Greenblatt, Pierre-Jacques J. Hamard, Takashi Asai, Na Man, Concepcion Martinez-Caja, Fan Liu, Stephen Nimer. Identification of CARM1/PRMT4 as a novel therapeutic target for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2017-3340

Background Particularly since the advent of lenalidomide, lower-risk myelodysplastic syndromes (MDS) patients with del(5q) have been the focus of many studies; however, the impact of age on disease characteristics and response to lenalidomide has not been analyzed. Methods We assessed the effect of age on clinical characteristics and outcomes in 286 lenalidomide-treated MDS patients with del(5q) from two multicenter trials. Results A total of 33.9, 34.3, and 31.8% patients were aged

Summary Epigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia., Highlights • L3MBTL1 is a chromatin-binding protein that represses SMAD5 expression • Lack of L3MBTL1 primes the hematopoietic development of pluripotent stem cells • L3MBTL1 regulates erythroid differentiation, Nimer, Perna, and colleagues report that the Polycomb protein L3MBTL1, deleted in 20q- myeloid malignancies, plays a regulatory role during the differentiation of human iPS cells. Upon lentiviral knockdown of L3MBTL1, iPS cells stay poised for hematopoietic development and upregulate BMP/SMAD target gene expression, leading to impaired neural development.

RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9–mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs.

Hodgkin's disease (HD) is a lymphoid malignancy characterized by the presence of Reed-Sternberg (RS) and Hodgkin's cells in a background of mixed inflammatory cells and stromal reaction. Studies have documented that HD is a neoplasm associated with abnormal cytokine and chemokine production. To define the expression of macrophage-derived chemokine (MDC) in HD, 57 cases (18 lymphocyte predominant, 11 mixed cellularity, 28 nodular sclerosis) were stained for MDC by immunohistochemistry and compared with reactive lymph nodes as controls. MDC was expressed by RS cells in classical HD (CHD) and showed a distinct cytoplasmic and Golgi localization. Accumulating evidence suggests that lymphocyte-predominant HD (LPHD) represents an entity distinct from CHD, with different biological properties and clinical course. On the basis of the high level of MDC staining alone, CHD could be distinguished from LPHD (P

The t(15;17) translocation in acute promyelocytic leukemia (APL) yields a PML/RAR-α fusion messenger RNA species that can be detected by reverse transcription–polymerase chain reaction (RT-PCR) amplification. Breakpoints within intron 3 of PML produce a short PML/RAR-α isoform, whereas breakpoints within intron 6 result in a longer form. Using RT-PCR, serial evaluations were performed on the bone marrow of 82 patients with APL (median follow-up, > 63 months) who received retinoic acid (RA) induction followed by postremission treatment with chemotherapy, RA, and biologic agents. Sixty-four patients attained a clinical complete remission and had at least 2 RT-PCR assays performed after completing therapy. Forty of 47 patients (85%) with newly diagnosed APL who were induced using RA had residual disease detectable by RT-PCR before additional therapy. After 3 cycles of consolidation therapy, residual disease was found in only 4 of 40 evaluable patients (10%). Among newly diagnosed patients who had 2 or more negative RT-PCR assays, only 3 of 41 (7%) had a relapse, whereas all 4 patients (100%) who had 2 or more positive results had a relapse. Among 63 newly diagnosed patients, those who expressed the short isoform appeared to have shorter disease-free and overall survival durations than patients who expressed the long isoform. These data indicate that 2 or more negative RT-PCR assays on bone marrow, performed at least 1 month apart after completing therapy, are strongly associated with long-term remissions. Conversely, a confirmed positive test is highly predictive of relapse.

MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G1 phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitro more efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G1. Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.

Deletions of the long arm of chromosome 20 have been reported in a wide range of myeloid disorders and may reflect loss of critical tumor suppressor gene(s). To identify such candidate genes, 65 human myeloid cell line DNAs were screened by polymerase chain reaction (PCR) for evidence of allelic loss at 39 highly polymorphic loci on the long arm of chromosome 20. A mono-allelic pattern was present in eight cell lines at multiple adjacent loci spanning the common deleted regions (CDRs) previously defined in primary hematological samples, suggesting loss of heterozygosity (LOH) at 20q. Fluorescence in situ hybridization (FISH) was then performed using a series of yeast artificial chromosomes (YACs) ordered in the CDR, and in five of eight cell lines, the deletions resulted from cytogenetically detectable whole chromosomal loss or large interstitial deletion, whereas in another cell line deletion was associated with an unbalanced translocation. LOH in the CMK megakaryocytic cell line, which has a hypotetraploid karyotype, was associated with a der(20)t(1;20)(q32;q12)x2 leading to complete deletion of the CDR. Three additional unbalanced translocations were found within the CDR and all three breakpoints mapped to a single YAC. We then used a series of P1 artificial chromosomes (PACs) spanning this YAC clone, and two PACs produced 'split' signals suggesting that they each span one of these breakpoints. Exon trapping using PACs that overlap the breakpoint regions yielded portions of six genes and evaluation of these genes as candidate tumor suppressor genes is underway. The limited information available about these genes suggests that the h-l(3)mbt gene is the most attractive candidate.

Background: Rearrangement between chromosome 8 and 21 is the most frequently observed chromosomal translocation. In AML patients, it leads to the formation of a novel fusion protein AML1-ETO. AML1-ETO functions as a transcriptional regulator to suppress myeloid differentiation and to promote self-renewal of hematopoiesis stem cells, which are of importance in leukemia development. However, AML1-ETO has no enzymatic function, thus targeting AML1-ETO directly is technically difficult. TAFII250, a largest subunit of the transcription factor IID complex (TFIID) serves to bring other components of basal transcription machinery to the preinitiation complex during the transcription initiation. And TAFII250 also plays a crucial role in the expression of genes involved in cell cycle and apoptosis. Yet despite the importance of these processes, there is no direct evidence implicating TAFII250 in cancer development. We have reported that acetylation of AML1-ETO on lysine 43 is critical for AML1-ETO mediated leukemogenesis and used a peptide pulldown strategy to identify proteins that preferring interact with acetylated AML1-ETO and identified TAFII250. Here, we are going to report the functions of TAFII250 in AML1-ETO induced leukemogenesis. Methods: We used Kasumi-1 and SKNO-1 cell lines derived from t(8; 21)AML patients, and a mouse AML1-ETO exon 9a (AE9a) expressing cell line developed from bone marrow cells of mice injected with bone marrow cells infected with AE9a to perform in vitro and in vivo studies. Results: We demonstrate that TAFII250 associates with AML1-ETO in AML cells through its bromodomain recognizing acetylated lysine 43 on AML1-ETO. The knockdown of TAFII250 completely abolished the proliferation of AML1-ETO expressing cells and has little influence on cell growth of non AML1-ETO expressing cells K562 and CD34+ cells. In addition, when we examined cleaved caspase 3 and PARP by western and measured annexin-V through flow cytometry, we found that the deficiency of TAFII250 induces apoptosis in AML1-ETO expressing cells. Further, the co-immunoprecipitation assay revealed that the deficiency of TAFII250 blocks the binding of AML1-ETO to CBFb (core binding factor beta subunit) and the recruitment of AML1-ETO at the promoter regions of a subset of its target genes. Consequently, loss of TAFII250 interferes with the expression of these genes. The depletion of TAFII250 also has negative effect on the self-renewal of leukemic cells, promoting their differentiation. Most importantly, the loss of TAFII250 severely impairs leukemia development in AE9a expressing cells. Conclusions: Together, these results reveal an essential role of TAFII250 in AML1-ETO mediated leukemogenesis and imply that TAFII250 might be a therapeutic target for AML1-ETO expressing AMLs. Disclosures No relevant conflicts of interest to declare.

Background: The FLT3-ITD (internal tandem duplication) is the most frequently observed molecular defect in acute myeloid leukemia (AML) and has been shown to be a poor prognostic factor in several studies. Many inhibitors of FLT3-ITD have been developed and tested in clinical trials; while a recent randomized study of PK412 (FLT3 inhibitor) showed significant clinical benefit in AML patients, many other studies have shown only modest effect. We and others have shown that the protein arginine methyltransferase 5 (PRMT5) is required to maintain self-renewal in many types of stem cells; we found that PRMT5 is expressed at much higher levels in hematopoietic stem cells than in differentiated cells and moreover, the expression of PRMT5 is increased in most acute myeloid leukemia cells and PRMT5 is also overexpressed in lymphoma and ovarian cancer cells. While we have been to define roles for PRMT5 in myeloproliferative disorders and normal hematopoietic stem and progenitor cells. The role of PRMT5 in AML has not been extensively studied. The mitogen activated protein kinases (MAPK) pathway is known to abnormally enhance the proliferation signal downstream of the FLT3-ITD. The dual-specificity mitogen activated protein kinase phosphatase-2 gene (DUSP4/MK-2) specifically dephosphorylates and inactivates MAPK. The role of DUSP4 in AML also is not clear. Here we reported that the role of PRMT5 and DUSP4 in AML with FLT3-ITD. Objective: The aim of the present study was to investigate whether the FLT3-ITD affects histone modification and/or the expression level of DUSP4 via effects on PRMT5 itself and also to explore whether the combination of a FLT3 inhibitor and a PRMT5 inhibitor could have synergistic anti-leukemic effects. Results: We showed that overexpression of FLT3-ITD increased the level of phospho-PRMT5 in 293T cells and that several FLT3 inhibitors reduced the phosphorylation of PRMT5 in MV4-11 cells. The FLT3-ITD binds PRMT5 when expressed in 293T cells and FLT3, FLT3-ITD, and FLT3-D835Y can directly phosphorylate PRMT5 in an in vitro kinase assay but this phosphorylation is inhibited by the AC220, in a dose-dependent fashion. Histone H2A arginine symmetric methylation, which is known to be a target of PRMT5, increases after treatment with CEP701 and AC220. These results suggest that the FLT3 inhibitor can enhance PRMT5 enzyme activity, and that FLT3-ITD inhibits PRMT5 enzymatic activity. We also showed that there was an enrichment of histone H2AR3/H4R symmetric di-methylation (sdm) at the promoter region of DUSP4. We also showed that absent PRMT5 increased the protein expression of DUSP4 and FLT3-ITD signaling suppression with AC220 decreased it through histone modification with Chip assay. We are able to show that PRMT5 inhibitor and sh-PRMT5 suppressed the cytokine signaling such as p-STAT5, p-Erk, and p-AKT that are known as signaling pathway that FLT3-ITD enhanced and DUSP4 suppressed. The PRMT5 was an essential gene for maintenance of MV4-11 cells. The sh-DUSP4 was able to partially rescue MV4-11 cells with treatment of sh-PRMT5. We showed that a FLT3 inhibitor and a PRMT5 inhibitor have synergistic anti-leukemic effects in MV4-11 cells. Conclusion: The FLT3-ITD affects the expression of DUSP4 through epigenetic modification such as the methylation status of histone H2AR3/H4R3 arginine symmetric methylation via PRMT5 in AML. The combination of an FLT3 inhibitor and PRMT5 inhibitor may have synergistic anti-leukemic effects in AML. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Background AML1-ETO (AE), a oncogenic protein generated by the t(8;21) translocation, causes acute myeloid leukemia (AML) in collaboration with other secondary events. The leukemogenicity of AE has been evaluated in multiple mouse models, such as expression of AE in Cdkn1a-null Hematopoietic stem cell (HSCs) and expression of AML1- ETO9a (AE9a), an alternatively spliced variant of AE, in WT HSCs. Both lead to the development of fully penetrant AML. Caspase-3 plays multiple roles in hematopoietic development and leukemia progression and treatment by affecting proliferation, self-renewal and differentiation. It has been shown that uncleaved caspase-3 levels are higher in the peripheral blood cells of AML patients compared to normal individuals, which suggests that the caspase pathway is dysregulated in AML. We and others have shown that Caspase-3 directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3 compromised background and accelerate leukemogenesis. Methods We developed a Caspase-3 knockout genetic mouse model of AML based on fetal liver cell transplantation. In brief, fetal liver cells from WT or Caspase3-/- mice were transduced to express AE9a in vitroand 100,000 AE9a+ transduced cells were transplanted into lethally irradiated recipient mice by tail-vein injection. Results We found loss of Caspase-3 impaired leukemia stem cell (LSC) self-renewal and delayed AE9a-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation or progression of AML. Moreover, we identified a new substrate of Caspase-3, ULK1, by in vitro cleavage assays and site-directed mutagenesis. ULK1 (serine/threonine UNC-51-like kinase) is the homology of Atg1 (the first autophagy related gene found in 1997) in mammalian cells, which is a direct target of mTOR and is responsible for initiation of the autophagic activity by forming a complex with mAtg13, FIP200 and Atg101. The induction of autophagy caused by upregulation of ULK1 in AE/AE9a-expressing Caspase-3-/- fetal liver cells acted to limit the leukemogenicity of AE9a in vivo. Inhibition of ULK1 by inhibitor or shRNAs could rescue the self-renewal capability induced by Caspase-3 deletion in serial replating assays. Unexpectedly, when we expressed AE/AE9a in fetal liver cells from WT and Caspase-3-/- mice, the protein levels were comparable suggesting the basal level of Caspase-3 didn't affect the expressing of AE/AE9a in fetal liver cells. Conclusion Autophagy may play a general role in the development and treatment of leukemia. In human AML, blasts display reduced expression of autophagy-related genes and decreased autophagic flux, indicating that low autophagy activity provides a general advantage for leukemia development. Beside this, a number of chemotherapy drugs have been reported to be able to induce leukemia cell death via activation of autophagy suggesting that autophagy plays critical roles in the leukemia treatment. Our study reveals that Caspase-3 regulates autophagy through its direct cleavage of ULK1 and this interaction dictates the pace of AE-driven leukemogenesis. Targeting this pathway may have therapeutic benefit for AML treatment. Disclosures No relevant conflicts of interest to declare.

Somatic mutations and chromosomal translocations of genes have emerged as major drivers in a range of hematopoietic malignancies. While ASXL1 is mutated in all forms of myeloid malignancies, ASXL2 is specifically mutated in t(8;21) AML patients. ASXL1 and ASXL2 mutations are mutually exclusive in t(8;21) AML. Despite the importance of ASXL2 mutations in clinical, it's role in leukemogenesis remain unknown. In the current study, we sought to dissect the role of ASXL2 in normal hematopoiesis and to identify the molecular mechanisms by which Asxl2 loss contributes to myeloid malignancies. In the current study, we utilized a mouse model of Asxl2 to characterize the hematopoietic features of in vivo. Asxl2-/- mice were characterized by pancytopenia and dysplastic features, including hyposegmented (bilobed) neutrophils with fine nuclear bridging (consistent with pseudo Pelger-Huët) and increased number of polychromatophilic red blood cells (RBCs), reminiscent of myelodysplastic syndrome (MDS). Flow cytometric analyses revealed that Asxl2-/- mice had an increased proportion of granulocytic/monocytic cells (Gr-1+/Mac1+) in the PB, BM and spleens compared to WT mice. The histologic analysis of the Asxl2+/- and Asxl2-/- spleen sections showed disrupted splenic architecture with an increased proportion of myeloid cells and massive accumulation of myeloperoxidase (MPO) positive cells in WT spleens. Asxl2-/- mice had an increased long-term (LH)-HSCs and granulocyte-macrophage progenitor (GMP) cells compared to WT mice.Consistently, the paired-daughter cell assays revealed that Asxl2-/- CD34-LSK BM cells had a higher proportion of cells with symmetric self-renewal capacity (SS, 62%) than WT cells (33%). In contrast, a significant reduction in the cells with symmetric differentiation potential was observed in Asxl2-/- HSCs (18%) compared to WT HSCs (40%), indicating a critical role of ASXL2 in the balance between the symmetric and asymmetric division of HSCs. Transplantation assays revealed that recipients transplanted with Asxl2-/- and Asxl2+/- bone marrow cells had shortened lifespan due to the development of MDS or AML, suggesting a cell-autonomous effect of Asxl2-loss in HSC/HPC functions. Furthermore, Asxl2-loss further increase the colony-forming potential and colony replating capacity of AML1-ETO expressing HSCs in vitro, suggesting a cooperative effect between AML1/ETO9a and Asxl2+/-to promote HSC self-renewal. RNA-seq analysis showed a unique signature of Asxl2-/- LK cells compared to WT LK cells. Gene set enrichment analysis revealed that altered expressed genes in Asxl2-/-LK cells were enriched in myeloid cell differentiation, hematopoiesis, apoptosis, and chromatin/nucleosome assembly signature. ChIP-seq analysis showed that differentially expressed genes were associated with dysregulated histone enhancer markers, including H3K27ac, H3K4me1, and H3K4me2. Further analysis demonstrated that the alteration of H3K27ac enrichment had a greater impact on gene expression, in comparison to H3K4me1/2. KEGG pathway analysis showed that genes with differential H3K27ac signals were enriched for hematopoietic cell lineage, cancer signaling pathway and myeloid leukemia development. IPA analysis further confirmed that genes with altered enrichment levels of were enriched in myeloid cell differentiation and apoptosis pathways. Altogether, these data suggest that ASXL2 regulates gene expression mainly through enhancer markers. Our results demonstrate that ASXL2 plays an important role in normal hematopoiesis, and Asxl2-loss in mice is sufficient to cause MDS-like disease and leukemia transformation. These results indicate that ASXL2 functions as a tumor suppressor in myelopoiesis. The Asxl2 knock-out mice present an ideal model for unveiling the mechanisms underlying the Asxl2-loss mediated multiple-step pathogenesis of myeloid malignancies and for testing novel therapeutic agents for myeloid malignant patients with ASXL2 alterations. Further studies to dissect the possible roles of ASXL2alterations in leukemogenesis and to identify therapeutic vulnerabilities they may create are ongoing. Disclosures No relevant conflicts of interest to declare.

Acute megakaryocytic leukemia is a rare subtype of AML that is often difficult to diagnose; it is most commonly associated with Down syndrome in children. To identify chromosomal imbalances and rearrangements associated with acute megakaryocytic leukemia, we used G-banding, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) on a variety of primary patients' samples and leukemia cell lines. The most common abnormality was gain of chromosome 19 or arm 19q, which was detected by CGH in four of 12 (33.3%) primary samples and nine of 11 (81.8%) cell lines. In none of the primary samples was this abnormality detected by G-banding analysis. WCP was used to define further the nature of the chromosome 19 gain in the cell lines, which was found to be due to the presence of additional 19q material on marker chromosomes or to cryptic translocations involving 19q. The most common chromosomal loss—detected only in the cell lines—was deletion of chromosomal band 13q14, which was seen in six of 11 (54.5%) cell lines. Other recurrent changes included gains of 1p, 6p, 8q, 11q, 15q, 17q, and 21q and losses of 2, 4q, 5q, 7q, 9p, and 11p. Combining conventional and molecular cytogenetic analyses defined recurrent clonal chromosomal abnormalities, which will aid in the identification of critical genes that are abnormal in acute megakaryocytic leukemia cells. © 2001 Wiley-Liss, Inc.

Innate immune signals and p53 contribute to the 5q– myelodysplastic syndrome, an acquired bone marrow failure disorder (pages 49–58 and 59–66).

The t(8;21) translocation, found in adult acute myelogenous leukemia, results in the formation of an AML1/ETO chimeric transcription factor. AML1/ETO expression leads to alterations in hematopoietic progenitor cell differentiation, although its role in leukemic transformation is not clear. The N-terminal portion of AML1, which is retained in AML1/ETO, contains a region of homology to the FAST proteins, which cooperate with Smads to regulate transforming growth factor beta1 (TGF-beta1) target genes. We have demonstrated the physical association of Smad proteins with AML1 and AML1/ETO by immunoprecipitation and have mapped the region of interaction to the runt homology domain in these AML1 proteins. Using confocal microscopy, we demonstrated that AML1, and ETO and/or AML1/ETO, colocalize with Smads in the nucleus of t(8;21)-positive Kasumi-1 cells, in the presence but not the absence of TGF-beta1. Using transient transfection assays and a reporter gene construct that contains both Smad and AML1 consensus binding sequences, we demonstrated that overexpression of AML1B cooperates with TGF-beta1 in stimulating reporter gene activity, whereas AML1/ETO represses basal promoter activity and blocks the response to TGF-beta1. Considering the critical role of TGF-beta1 in the growth and differentiation of hematopoietic cells, interference with TGF-beta1 signaling by AML1/ETO may contribute to leukemogenesis.

High-dose chemoradiotherapy (HDT) with autologous stem cell transplantation (ASCT) is the treatment of choice for patients with relapsed aggressive non-Hodgkin lymphoma (NHL). However, its role in the treatment of patients with primary refractory disease is not well defined. The outcomes of 85 patients with primary refractory aggressive NHL who underwent second-line chemotherapy with ICE with the intent of administering HDT/ASCT to those patients with chemosensitive disease were reviewed. Patients were retrospectively classified as induction partial responders (IPR) if they attained a partial response to doxorubicin-based front-line therapy or as induction failures (IF) if they had less than partial response. Forty-three patients (50.6%) had ICE-chemosensitive disease; there was no difference in the response rate between the IPR and the IF groups. Intention-to-treat analysis revealed that 25% of the patients were alive and 21.9% were event-free at a median follow-up of 35 months. Among 42 patients who underwent transplantation, the 3-year overall and event-free survival rates were 52.5% and 44.2%, respectively, similar to the outcomes for patients with chemosensitive relapsed disease. No differences were observed between the IPR and IF groups, and there were no transplantation-related deaths. More than one extranodal site of disease and a second-line age-adjusted International Prognostic Index of 3 or 4 before ICE chemotherapy were predictive of poor survival. These results suggest that patients with primary refractory aggressive NHL should receive second-line chemotherapy, with the intent of administering HDT/ASCT to those with chemosensitive disease. Newer therapies are needed to improve the outcomes of patients with poor-risk primary refractory disease.

Identification and characterization of leukemia-related chromosomal translocations have had significant impact on all aspects of the management of acute leukemia, including its diagnosis, assignment of prognosis, and development of an appropriate treatment plan. Several genes are recurrent targets of chromosomal abnormalities, suggesting that they play a key role in leukemogenesis. Significant progress has been made to define potentially unifying molecular mechanisms of leukemic transformation. Hopefully, these findings will provide the basis for molecularly targeted therapies for leukemia.

The AML1 and ETS families of transcription factors play critical roles in hematopoiesis; AML1, and its non-DNA-binding heterodimer partner CBFbeta, are essential for the development of definitive hematopoiesis in mice, whereas the absence of certain ETS proteins creates specific defects in lymphopoiesis or myelopoiesis. The promoter activities of numerous genes expressed in hematopoietic cells are regulated by AML1 proteins or ETS proteins. MEF (for myeloid ELF-1-like factor) is a recently cloned ETS family member that, like AML1B, can strongly transactivate several of these promoters, which led us to examine whether MEF functionally or physically interacts with AML1 proteins. In this study, we demonstrate direct interactions between MEF and AML1 proteins, including the AML1/ETO fusion protein, in t(8;21)-positive acute myeloid leukemia (AML) cells. Using mutational analysis, we identified a novel ETS-interacting subdomain (EID) in the C-terminal portion of the Runt homology domain (RHD) in AML1 proteins and determined that the N-terminal region of MEF was responsible for its interaction with AML1. MEF and AML1B synergistically transactivated an interleukin 3 promoter reporter gene construct, yet the activating activity of MEF was abolished when MEF was coexpressed with AML1/ETO. The repression by AML1/ETO was independent of DNA binding but depended on its ability to interact with MEF, suggesting that AML1/ETO can repress genes not normally regulated by AML1 via protein-protein interactions. Interference with MEF function by AML1/ETO may lead to dysregulation of genes important for myeloid differentiation, thereby contributing to the pathogenesis of t(8;21) AML.

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal–induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal–induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal–induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal–induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.

The 8;21 translocation is the most common cytogenetic abnormality in human acute myelogenous leukemia, joining the AML1 gene on chromosome 21, to the ETO gene on chromosome 8, forming the AML1/ETO fusion gene. The AMLI/ETO fusion protein has been shown to function mainly as a transcriptional repressor of AML1 target genes and to block AML1 function in vitro and in vivo. However, AML1/ETO can also activate the BCL-2 promoter and cause enhanced hematopoietic progenitor self-renewal in vitro, suggesting gain-of-functions unique to the fusion protein. We used NIH3T3 cells to determine the transforming capacity of AML1/ETO, and to further characterize its mechanism of action. Expression of AML1/ETO in NIH3T3 cells caused cell-type specific cell death, and cellular transformation, characterized by phenotypic changes, anchorage-independent growth, and tumor formation in nude mice. In contrast, neither expression of AML1A, AML1B or ETO altered the normal growth pattern of the cells. To investigate the mechanism of transformation by AML1/ETO, we analysed the levels of activated, phosphorylated c-Jun (ser63) and other constituents of the AP-1 complex, in the presence of various AML1/ETO related proteins. Expression of AML1/ETO increased the level of c-Jun-P (ser63), and activated AP-1 dependent transcription, which was inhibited by expression of a dominant-negative c-Jun protein. Mutational analysis revealed that the runt homology domain (RHD) and a C-terminal transcriptional repression domain in AML1/ETO are required for transformation, activation of c-Jun and increased AP-1 activity. These results establish the transforming potential of the t(8;21) fusion protein and link this gain-of-function property to modulation of AP-1 activity.

We analyzed a group of 51 patients with primary refractory and relapsed intermediate-grade lymphoma (IGL) from the time of initiation of three cycles of second-line chemotherapy, ifosfamide, carboplatin and etoposide (ICE), in whom the intent was to administer curative high-dose chemoradiotherapy and autologous stem cell transplantation (ASCT). We sought to determine if the International Prognostic Index (IPI) assessed immediately prior to ICE, second-line IPI (sIPI), was predictive of outcome. The response rate to ICE-based chemotherapy was 69%, and 47% of the transplanted patients remain failure-free at 2.5 years. Stratification of patients based upon the sIPI demonstrated a superior 2.5 year failure-free survival (FFS) curve for patients with low (I) or low-intermediate (II) risk disease vs. those with high-intermediate (III) and high (IV) risk disease (45% vs. 9%, P

A cytogenetically cryptic (12;21) translocation is the most common molecular abnormality identified in childhood acute lymphoblastic leukemia (ALL), and it generates a chimeric TEL-AML1 protein. Fusion of the Helix-Loop-Helix (HLH) (also called the pointed) domain of TEL to AML1 has been suggested to convert AML1 from a transcriptional activator to a repressor. To define the structural features of this chimeric protein required for repression, we analysed the transcriptional activity of a series of TEL-AML1 mutants on the AML1-responsive interleukin-3 (IL-3) promoter, a potentially relevant gene target. Our results demonstrate that TEL-AML1 represses basal IL-3 promoter activity in lymphoid cells, and deletion mutant analysis identified three distinct domains of TEL-AML1 that are required for repression; the HLH (pointed) motif contained in the TEL portion of TEL-AML1, and both the runt homology domain (Rhd) and the 74 amino acids downstream of the Rhd that are present in the AML1 portion of the fusion protein. Although AML1B (and a shorter AML1 isoform, AML1A) have transcriptional activating activity on the IL-3 promoter, fusion of the AML1 gene to the TEL gene generates a repressor of IL-3 expression. Consistent with this activity, freshly isolated human ALL cells that contain TEL-AML1 do not express IL-3.

In up to 30% of acute myeloid leukemia (AML), chromosomal translocations disrupt one of the core binding factor (CBF) genes most frequently by the t(8;21)(q22;q22). Although patients with CBF leukemia generally have a higher complete remission rate and disease-free survival as compared to those with a normal karyotype or other chromosomal aberrations, additional distinct mutations in genes involved in signal-transduction pathways, particularly in receptor tyrosine kinases (RTKs), herald a higher relapse rate (see below).

Hepatitis-associated aplastic anemia (HAAA) is an uncommon disorder that usually is not due to hepatitis A or B virus infection. Hepatitis C virus (HCV) seropositivity is infrequently observed in aplastic anemia (AA) patients who have not been extensively transfused. However, HCV seropositivity may not be detected until several weeks or months after viral infection and AA patients may exhibit defective humoral immunity. Therefore, we evaluated sera from AA patients for the presence of HCV viremia using a reverse transcriptase polymerase chain reaction (RT-PCR) based assay and several serologic assays for HCV antibodies. Serum samples from 90 AA patients who presented to the UCLA Medical Center between March 1984 and February 1990 were analyzed. Overall, 17 patients were found to have HCV viremia by RT-PCR assay, of whom 14 had a positive second-generation HCV enzyme immunoassay (EIA-2) and only 6 were EIA-1 reactive. The frequency of HCV viremia increased with the duration of time between diagnosis and sample procurement, and the number of blood products transfused prior to sampling (P = 0.026). No patient who received fewer than 20 U of blood products or who was sampled less than 20 days after diagnosis had a positive HCV RT-PCR result. Of four patients with hepatitis-associated AA (HAAA), one who was sampled 23 days after diagnosis had hepatitis C viremia and a reactive EIA-2 assay. Therefore, the high frequency of HCV viremia in this patient population is most likely due to transfusion with contaminated blood products prior to the introduction of routine blood donor screening for HCV.

To examine the safety and efficacy of recombinant-methionyl human stem cell factor (r-metHuSCF), 38 patients with intermediate-grade or immunoblastic high-grade non-Hodgkin's lymphoma who were eligible for autologous transplantation were randomized to receive r-metHuSCF (5, 10, 15, or 20 μg/kg/d) plus Filgrastim (10 μg/kg/d) or Filgrastim (10 μg/kg/d) alone to mobilize peripheral blood progenitor cells. Subcutaneous administration of r-metHuSCF was well tolerated in conjunction with a multi-agent pre-medication regimen; local injection site reactions were the most commonly seen adverse event. The total mononuclear cell count, CD34+ cell content, granulocyte-macrophage colony-forming cells (GM-CFC), and burst-forming units-erythroid (BFU-E) per kilogram in the apheresis product was similar when all patients were analyzed by treatment cohort and mobilization regimen (Filgrastim or r-metHuSCF in combination with Filgrastim); however, when prior chemotherapy was taken into account in a supplementary analysis, clinically important differences were observed. Extensive prior therapy was defined as the amount of exposure to specific stem cell toxic chemotherapeutic agents that patients received. These agents include procarbazine, nitrogen mustard, melphalan, nitrosoureas (≥2 cycles of any of these drugs) or greater than 7.5 g of cytosine arabinoside. In these patients, there was an increased number of CD34+ cells (1.76 v 0.28 × 106/kg), GM-CFC (20.5 v 5.0 × 104/kg), and BFU-E (36.9 v 8.9 × 104/kg) in patients receiving r-metHuSCF and Filgrastim (N = 18) compared with Filgrastim alone (N = 5). These patients also had a decreased time to an untransfused platelet count of 20 × 109/L that was 10.5 days shorter in the patients who received r-metHuSCF and Filgrastim (12.5 v 23 days). These differences were not found to be statistically significant, possibly because of small size, but are clinically important.

The E2A-HLF (hepatic leukemia factor) oncoprotein, generated in pro-B lymphocytes by fusion of the trans-activation domain ofE2Ato the basic region/leucine zipper (bZIP) domain ofHLF,functions as an anti-apoptotic transcription factor in leukemic cell transformation. When introduced into interleukin 3 (IL-3)-dependent mouse pro-B lymphocytes, E2A-HLF prevents apoptosis induced by growth factor deprivation, suggesting that IL-3 mediates cell survival through activation of a transcription factor whose activity can be constitutively replaced by the chimeric oncoprotein. We considered four bZIP transcription factors as candidates for this putative IL-3-regulated factor, each of which binds avidly to the DNA consensus sequence recognized by E2A-HLF and is related to theCaenorhabditis elegansCES-2 (cell death specification protein) neuron-specific mediator of cell death. The expression and binding activity of the Nfil3 protein (also called E4bp4), but not of Hlf, Dbp, or Tef, was found to be regulated by IL-3 in mouse pro-B cell lines (Baf-3 and FL5.12). Northern blot analysis showed thatNfil3/E4bp4is regulated as a “delayed-early” IL-3-responsive gene, requiringde novoprotein synthesis. In the absence of IL-3, enforced expression of the humanNFIL3/E4BP4cDNA promoted the survival but not the growth of IL-3-dependent pro-B cells. Our results implicate NFIL3/E4BP4 (nuclear factor regulated by IL-3/adenovirus E4 promoter binding protein) in a distinct growth factor-regulated signaling pathway that is responsible for the survival of early B-cell progenitors, and whose alteration by E2A-HLF leads to childhood B lineage leukemia.

A number of prognostic factors affect outcome in patients with relapsed or primary refractory diffuse large B-cell lymphoma (DLBCL), including refractory disease and the second-line age-adjusted international prognostic index. In de novo DLBCL, the cell of orgin, as determined by expression microarray analysis or immunohistochemistry (IHC), predicts event-free survival (EFS). We evaluated the cell of origin, as well as other pathologic markers of outcome, on the repeat biopsy specimen of 88 transplantation-eligible patients undergoing ifosfamide, carboplatin, etoposide (ICE) second-line chemotherapy (SLT) followed by high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) to see if were they prognostic in the salvage setting. Pretreatment clinical factors were well balanced between the cohorts. There was no significant difference in response to SLT, HDT, event-free or overall survival based on the cell of origin or any of the common pathologic markers examined. The cell of origin as determined by IHC does not predict outcome in transplantation-eligible patients with relapsed or primary refractory DLBCL.

Hematopoiesis is a tightly regulated process involving the control of gene expression that directs the transition from hematopoietic stem and progenitor cells to terminally differentiated blood cells. In leukemia, the processes directing self-renewal, differentiation, and progenitor cell expansion are disrupted, leading to the accumulation of immature, non-functioning malignant cells. Insights into these processes have come in stages, based upon technological advances in genetic analyses, bioinformatics, and biological sciences. The first cytogenetic studies of leukemic cells identified chromosomal translocations that generate oncogenic fusion proteins, and most commonly affect regulators of transcription. This was followed by the discovery of recurrent somatic mutations in genes encoding regulators of the signal transduction pathways that control cell proliferation and survival. Recently, studies of global changes in methylation and gene expression have led to the understanding that the output of transcriptional regulators and the proliferative signaling pathways, are ultimately influenced by chromatin structure. Candidate gene, whole genome, and whole exome sequencing studies have identified recurrent somatic mutations in genes encoding epigenetic modifiers in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In contrast to the two hit model of leukemogenesis, emerging evidence suggests that these epigenetic modifiers represent a class of mutations that are critical to the development of leukemia and affect the regulation of various other oncogenic pathways. In this review, we discuss the range of recurrent, somatic mutations in epigenetic modifiers found in leukemia and how these modifiers relate to the classical leukemogenic pathways that lead to impaired cell differentiation and aberrant self-renewal and proliferation.

Necdin, a member of the melanoma antigen gene (MAGE) family, originally identified to suppress cell proliferation in post-mitotic neurons, facilitates the entry of the cell into cell cycle, functions like retinoblastoma (Rb) protein, and acts as a transcriptional repressor that recognizes guanosine (G)-rich DNA sequences. Necdin also binds to p53 and inhibits p53-dependent apoptosis in these cells, demonstrating its function as an endogenous anti-mitotic and anti-apoptotic protein in post-mitotic neurons [1]. The necdin gene is located on chromosome 15 in human and its expression is controlled through genomic imprinting; it is one of the genes that have been associated with the human Prader-Willi syndrome, a genetic neurobehavioral disorder [2]. Necdin is expressed in some stem or undifferentiated progenitor cells such as mesoangioblast stem cells [3], vessel-associated stem cells [4], adipocyte progenitor cells [5, 6], and hematopoietic stem cells (HSCs) [7], yet the function of necdin in these cells remains unclear. While highly expressed in long-term HSCs, transcript profiling of hematopoietic stem/progenitor cells (HSPCs) isolated from wild-type and p53 null mice identified necdin as being a p53 target gene [7]. We then demonstrated using chromatin immunoprecipitation assays that the necdin promoter contains a p53 binding site and that necdin is a direct transcriptional activator of p53 in HSPCs. Lentivirus-mediated knock down or overexpression of necdin resulted in decreased or increased HSPC quiescence, respectively [7]. We recently clarified the function of necdin in HSCs and HSPCs using necdin null mice [8]. Necdin null mice show the same phenotype as the patients with Prader-Willi syndrome and because they die perinatally, we transplanted necdin null fetal liver cells into lethally irradiated wild type recipients to reconstitute their bone marrow function and analyzed the HSC intrinsic function of necdin in adult hematopoiesis. Necdin-null HSCs have normal self-renewal capacity after serial transplantation, while we found that necdin-null HSCs are less quiescent and more proliferative than normal, suggesting that necdin has the similar function as p53 in promoting HSC quiescence in the steady state. We then demonstrated that necdin-null HSCs are highly sensitive to genotoxic stress, irradiation or chemotherapy, with increased apoptosis via both cell-cycle-dependent and cell-cycle independent mechanisms. We further found that the function of necdin in hematopoiesis in response to genotoxic stress depends on Gas2L3, a novel member of the Gas2 family, using

Therapy-related myelodysplastic syndromes and acute myelogenous leukemia comprise a poor-risk subset of myelodysplastic syndromes and acute myelogenous leukemia. Large-scale mutation profiling efforts in de novo myelodysplastic syndromes have identified mutations that correlate with clinical features, but such mutations have not been investigated in therapy-related myelodysplastic syndromes and acute myelogenous leukemia. Genomic DNA from 38 patient samples were subjected to high throughput polymerase chain reaction and sequenced for TP53, TET2, DNMT3A, ASXL1, IDH1, IDH2, EZH2, EED, SUZ12, RBBP4, SRSF2, U2AF35, and SF3B1. We identified somatic mutations in 16 of 38 (42%) patients. TP53 mutations were the most common lesion, detected in 8 of 38 (21%) patients, followed by TET2 in 4 of 38 (10.5%). Cases with a TP53 mutation or loss of the TP53 locus had a worse overall survival compared to those with wild-type TP53 (8.8 vs. 37.4 months; P=0.0035).

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis.

We hypothesized that the high early death rate (EDR) due to bleeding in acute promyelocytic leukemia (APL) is in part attributable to delays in all- trans retinoic acid (ATRA). We conducted a retrospective analysis of the timing of ATRA administration. 204 consecutive patients with newly diagnosed APL between 1992 and 2009 were identified. The EDR was 11%. 44% of early deaths occurred in the first week. Hemorrhage accounted for 61% of early deaths. ATRA was ordered the day APL was suspected in 31% of patients. Delays in ATRA administration led to increases in the percentage of early deaths from hemorrhage.

The clinical heterogeneity of patients with myelodysplastic syndromes suggests that there must also be a broad range of pathogenetic abnormalities that underlie this disorder. It is clear that the molecular abnormalities associated with MDS are vast, as are the functional abnormalities within the hematopoietic compartment. This implies that there may also be significant differences in the cell of origin and varying degrees of aberrant communication with the microenvironment in individual patients. It is only through recent advances in flow cytometry, DNA sequencing, and high throughput analysis of the genome, proteome, and RNA expression profiles that we can make these general but profound statements about the spectrum of abnormalities in MDS patients.

Both copies of a repeated sequence CATT(A/T), located between bp -53 and -39 in the upstream region of the human GM-CSF gene, are required for mitogen-inducible promoter activity in T lymphocytes. However, the proteins that recognize this region of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, and are responsible for its transcriptional regulatory activity, have not been clearly identified. Using transient transfection assays, we demonstrate that a 19-bp oligonucleotide containing the CATT(A/T) repeats has strong constitutive enhancer activity in both T cell and non-T-cell lines, even though GM-CSF is not normally constitutively expressed by these cells. A 12-bp oligonucleotide, containing only the sequence CATTAATCATTT, lacks enhancer activity indicating that the nucleotides surrounding these sequences are critical for this enhancer activity. The sequence TTTCCT, which can bind members of the ets family of transcription factors, is located just 3′ of these CATT(A/T) repeats, and mutagenesis of the CCT sequence abolishes (1) the constitutive (and mitogen inducible) enhancer activity of the 19-bp GM-CSF sequences, (2) the responsiveness to transactivation by ets-1, and (3) the ability to specifically bind ets-1 and elf-1 in electrophoretic mobility shift assays (EMSA). We demonstrate that although T cells contain nuclear proteins capable of independently recognizing the ets binding site and the CATT(A/T) repeats in EMSAs, both of these regulatory elements are required for enhancer function. The strong constitutive activity of this 19-bp region suggests that negative regulation of the GM-CSF promoter is critical for the restricted expression pattern of GM-CSF mRNA.

To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a lambda gt11 cDNA library, constructed from phytohemagglutinin-stimulated human T-cell RNA, with a probe containing regulatory sequences in the upstream region of the IL-3 gene (located from bp -165 to -128 and referred to as the DNase I footprint A region). We isolated a 0.96-kb cDNA that encoded a basic amino acid domain and a leucine zipper domain and used the "rapid amplification and cloning of 3' ends" technique to isolate the 3' half of the cDNA clone, generating a 1.9-kb full-length cDNA clone. Using in vitro-translated protein, which we call NF-IL3A, we defined the IL-3 promoter sequences bound by NF-IL3A in DNase I footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence required for binding of NF-IL3A in vitro. Proteins that bind to the NF-IL3A binding site are found in both unstimulated and stimulated T-cell lines in similar amounts, although the level of NF-IL3A mRNA increases after T-cell activation in several mature T-cell lines. The NF-IL3A protein is nearly identical to a recently identified transcriptional repressor protein, E4BP4, and NF-IL3A binds specifically to regulatory sequences in both the adenovirus E4 promoter and the human gamma interferon promoter. Cotransfection experiments demonstrate that introduction of an expression vector containing the NF-IL3A cDNA into resting T cells transactivates IL-3 promoter-chloramphenicol acetyltransferase gene plasmids that contain the A region; this effect requires the presence of an intact NF-IL3A binding site. One or more copies of the A region also confer NF-IL3A responsiveness on a heterologous promoter in T cells. NF-IL3A appears to play an important role in the expression of IL-3 by T cells.