The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis.

The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.

Fludarabine, melphalan, and alemtuzumab conditioning in adults with standard-risk advanced acute myeloid leukemia and myelodysplastic syndrome.

PURPOSE: This prospective phase II study evaluated toxicity, relapse rate, progression-free survival, and overall survival after allogeneic transplantation and conditioning with fludarabine, melphalan, and alemtuzumab in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). PATIENTS AND METHODS: Fifty-two consecutive adults with AML and MDS were enrolled onto the study. Median age was 52 years (range, 17 to 71 years) and the majority of patients had high-risk disease, comorbidities, and/or modest reduction in performance status. Fifty-six percent of patients had unrelated or mismatched related donors. RESULTS: After a median follow-up of 18 months (range, 2 to 34 months), 1-year survival was 48% (95% CI, 34% to 61%), progression-free survival was 38% (95% CI, 25% to 52%), relapse rate was 27% (95% CI, 15% to 40%), and treatment-related mortality was 33% (95% CI, 20% to 46%). The cumulative probability of extensive chronic graft-versus-host disease (GVHD) was only 18% (95% CI, 8% to 40%); extensive chronic GVHD was only observed in recipients of unrelated donor transplants. Performance score and disease status were the major predictors of outcome. High-risk disease (ie, active AML or MDS with > 5% blasts) or even modest decreases in performance status were associated with poor outcomes. Patients with standard-risk leukemia (first or second complete remission) or MDS (< 5% blasts) had excellent outcomes despite unfavorable disease characteristics. CONCLUSION: Fludarabine and melphalan combined with in vivo alemtuzumab is a promising transplantation regimen for patients with AML or MDS and low tumor burden. For patients with active disease, this regimen provides at best modest palliation. Despite a low incidence of GVHD, transplantation is still associated with considerable nonrelapse mortality in patients with decreased performance status.

Detection of 11q23/MLL rearrangements in infant leukemias with fluorescence in situ hybridization and molecular analysis.

Cytogenetic abnormalities of band 11q23 have been found in more than 50% of infant leukemias regardless of the phenotype. Using probes for the MLL gene at 11q23, MLL rearrangements have been identified in 70-80% of all infant leukemias including virtually all of the cases with 11q23 translocations, as well as cases with apparently normal karyotypes. We reviewed the chromosomal pattern of 26 cases of infant leukemias (12 ALL, 12 AML, two AUL). Eleven had 11q23 translocations, five had other abnormalities, and 10 had a normal karyotype. To determine whether 11q23/MLL rearrangements were present in the leukemia cells of patients with a normal karyotype, we performed FISH and molecular studies of eight of these patients who had adequate material. Three were found to have 11q23/MLL abnormalities, two of them detected by FISH; one ALL case had a t(11;19) (q23;p13.3), and one AML case had a t(11;19) (q23;p13.1). Retrospective review confirmed the presence of the t(11;19) in a small percentage of poor quality metaphase cells in both cases. A rearrangement of the MLL gene was detected by Southern blot analysis of leukemic cells from a third patient with ALL; one cell with a deletion of 11q23 was found on karyotypic review. Therefore, in our series the actual incidence of 11q23 abnormalities in infant leukemias was 54% (14/26): 67% in ALL (8/12) and 50% in AML (6/12). Our findings suggest that most infant leukemias with apparently normal karyotypes that have a molecular rearrangement of the MLL gene are undetected subtle translocations.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of MLL gene rearrangements in adult acute lymphoblastic leukemia. A Cancer and Leukemia Group B study.

Specific structural rearrangements involving chromosome band 11q23 occur in a variety of hematologic malignancies, including an estimated 2-7% of patients with acute lymphoblastic leukemia (ALL). Translocations involving chromosome band 11q23 have been associated with a poor prognosis in patients with ALL. Recently, a gene known as MLL has been identified which is involved in acute lymphoid and myeloid leukemias with rearrangements at 11q23. A 0.74-kilobase (kb) cDNA probe from the MLL gene can detect both common and uncommon rearrangements involving MLL on conventional Southern blots. We studied 86 newly diagnosed adults entered on an ALL clinical trial to investigate the incidence of MLL gene rearrangements and to determine clinical, morphologic, immunologic and cytogenetic characteristics of such patients. Two of 86 patients had MLL gene rearrangements detected by Southern blot analysis. One of these 86 patients had an 11q23 translocation by cytogenetic analysis whereas the second patient was unevaluable by standard cytogenetic analysis. Southern blot identification of rearrangements involving MLL, especially in patients with limited material for cytogenetic analysis, can provide critical diagnostic and prognostic information which may be useful in the clinical management of patients with these abnormalities.

Bronchiolitis obliterans organizing pneumonia as a complication of allogeneic bone marrow transplantation.

We report a patient who underwent two allogeneic bone marrow transplants for chronic myelogenous leukemia, initially in 1984 and again after relapse in 1990, who developed an identical pulmonary syndrome at a similar interval following each transplant. The patient presented with a non-productive cough, bilateral inspiratory crackles, and multiple patchy infiltrates on chest X-ray. Pulmonary function testing revealed a restrictive abnormality but no obstructive defects. The appearance of this pulmonary disorder after each transplant coincided with the development of chronic graft-versus-host disease. In both instances, this pulmonary syndrome completely reversed with corticosteroid therapy. The patient's chest computed tomographic scan and lung biopsy specimens were consistent with the diagnosis of bronchiolitis obliterans with organizing pneumonia (BOOP). While bronchiolitis obliterans has been reported following allogeneic transplant, BOOP has not previously been reported in this setting.

The human MLL gene: nucleotide sequence, homology to the Drosophila trx zinc-finger domain, and alternative splicing.

We have previously reported the cloning of several cDNAs corresponding to the MLL gene. The predicted primary amino acid sequence of two of these clones, 14p-18B and 14-7, reveals nearly complete identity with parts of the sequences of HRX, ALL-1, and Htrx-1, including a Zinc-finger region with homology to the Drosophila trithorax gene. However, we found that there is a stretch of 39 amino acids that is absent from 14p-18B when compared to ALL-1 and HRX. Another sequence of three amino acids is present in ALL-1, but is absent from 14p-18B and HRX. Nucleotide sequence examination reveals that these differences arise from alternative splicing, suggesting that MLL, HRX, and ALL-1 each represents a different alternative splicing product from the same gene. At least two cDNA clones, 14-7 and 14p-18C, correspond to incompletely processed transcripts including intron sequences. Northern blots using a subclone of 14p-18B revealed mRNA species of 14-16 kb in size in various human tissues. RNase protection assays show that the splice variant containing exon 8 and lacking a 9-bp extension 3' of exon 12 is predominantly expressed in hematopoietic cell lines.

Therapy-related myeloid leukemia.

One of the most serious possible consequences of cancer therapy is the development of a second cancer, especially leukemia. Several distinct subsets of therapy-related leukemia can be distinguished currently. These include classic therapy-related myeloid leukemia, leukemia that follows treatment with agents that inhibit topoisomerase II, acute lymphoblastic leukemia, and leukemias with 21q22 rearrangements or inv(16) or t(15;17). These types of leukemia are discussed in detail in this article.

Regulation of MEIS1 by distal enhancer elements in acute leukemia.

Aberrant activation of the three-amino-acid-loop extension homeobox gene MEIS1 shortens the latency and accelerates the onset and progression of acute leukemia, yet the molecular mechanism underlying persistent activation of the MEIS1 gene in leukemia remains poorly understood. Here we used a combined comparative genomics analysis and an in vivo transgenic zebrafish assay to identify six regulatory DNA elements that are able to direct green fluorescent protein expression in a spatiotemporal manner during zebrafish embryonic hematopoiesis. Analysis of chromatin characteristics and regulatory signatures suggests that many of these predicted elements are potential enhancers in mammalian hematopoiesis. Strikingly, one of the enhancer elements (E9) is a frequent integration site in retroviral-induced mouse acute leukemia. The genomic region corresponding to enhancer E9 is differentially marked by H3K4 monomethylation and H3K27 acetylation, hallmarks of active enhancers, in multiple leukemia cell lines. Decreased enrichment of these histone marks is associated with downregulation of MEIS1 expression during hematopoietic differentiation. Further, MEIS1/HOXA9 transactivate this enhancer via a conserved binding motif in vitro, and participate in an autoregulatory loop that modulates MEIS1 expression in vivo. Our results suggest that an intronic enhancer regulates the expression of MEIS1 in hematopoiesis and contributes to its aberrant expression in acute leukemia.

A novel clofarabine bridge strategy facilitates allogeneic transplantation in patients with relapsed/refractory leukemia and high-risk myelodysplastic syndromes.

Patients with relapsed/refractory leukemias or advanced myelodysplastic syndrome (MDS) fare poorly following allogeneic hematopoietic cell transplant (HCT). We report prospective phase II study results of 29 patients given clofarabine 30 mg/m(2)/day i.v. × 5 days followed immediately by HCT conditioning while at the cytopenic nadir. A total of 15/29 patients (52%) were cytoreduced according to pre-defined criteria (cellularity <20% and blasts <10%). Marrow cellularity (P<0.0001) and blast% (P=0.03) were reduced. Toxicities were acceptable, with transient hyperbilirubinemia (48%) and gr3-4 infections (10%). In all, 28/29 proceeded to transplant; 27 received ATG or alemtuzumab. Post HCT, 180 day non-relapse mortality (NRM) was 7% (95% confidence interval (CI): 1-21), relapse was 29% (95% CI: 13-46) and OS was 71% (95% CI: 51-85), comparing favorably to published data for high-risk patients. Two-year graft vs host disease incidence was 40% (95% CI: 21-58) and 2 year OS was 31% (95% CI: 14-48). Disease at the nadir correlated with inferior OS after HCT (HR=1.22 for each 10% marrow blasts, 95% CI: 1.02-1.46). For AML/MDS patients, there was a suggestion that successful cytoreduction increased PFS (330 vs 171 days, P=0.3) and OS (375 vs 195 days, P=0.31). Clofarabine used as a bridge to HCT reduces disease burden, is well tolerated, and permits high-risk patients to undergo HCT with acceptable NRM. Late relapses are common; thus, additional strategies should be pursued. NCT-00724009.

Neurotoxicity of meperidine.

Meperidine neurotoxicity manifests as shakiness, tremors, myoclonus, and seizures. It is generally seen with repeated parenteral use. We report a case of meperidine neurotoxicity from oral use by an otherwise healthy woman. The pharmacology and clinical implications are discussed.

U937 cell line has a t(10;11)(p13-14;q14-21) rather than a deletion of 11q.

The U937 cell line was studied with the fluorescence in situ hybridization (FISH) technique using phage and cosmid probes which were mapped and ordered on 11q. Although this cell line was thought to have a del(11q), FISH demonstrated that 11q was translocated to 10p and that the breakpoint on 11q is centromeric to the MLL gene. This 10;11 translocation appears to be a t(10;11)(p13-14;q14-21), which was recently reported to be a recurring translocation in malignant hematologic disease. This cell line will be a good tool for the study of this chromosomal rearrangement.

Chromatin-related properties of CBP fused to MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia.

As a result of the recurring translocation t(11;16) (q23;p13.3), MLL (mixed-lineage leukemia) is fused in frame to CBP (CREB binding protein). This translocation has been documented almost exclusively in cases of acute leukemia or myelodysplasia secondary to therapy with drugs that target DNA topo isomerase II. The minimal chimeric protein that is produced fuses MLL to the bromodomain, histone acetyltransferase (HAT) domain, EIA-binding domain and steroid-receptor coactivator binding domains of CBP. We show that transplantation of bone marrow retrovirally transduced with MLL-CBP induces myeloid leukemias in mice that are preceded by a long preleukemic phase similar to the myelodysplastic syndrome (MDS) seen in many t(11;16) patients but unusual for other MLL translocations. Structure-function analysis demonstrated that fusion of both the bromodomain and HAT domain of CBP to the amino portion of MLL is required for full in vitro transformation and is sufficient to induce the leukemic phenotype in vivo. This suggests that the leukemic effect of MLL-CBP results from the fusion of the chromatin association and modifying activities of CBP with the DNA binding activities of MLL.

Retrovirus-mediated gene transfer of MLL-ELL transforms primary myeloid progenitors and causes acute myeloid leukemias in mice.

The MLL-ELL fusion gene results from the translocation t(11;19)(q23;p13.1) that is associated with de novo and therapy-related acute myeloid leukemia. To study its transforming properties, we retrovirally transduced primary murine hematopoietic progenitors and assessed their growth properties both in vitro and in vivo. MLL-ELL increased the proliferation of myeloid colony-forming cells in methylcellulose cultures upon serial replating, whereas overexpression of ELL alone had no effect. We reconstituted lethally irradiated congenic mice with bone marrow progenitors transduced with MLL-ELL or the control MIE vector encoding the enhanced green fluorescent protein. When the peripheral blood of the mice was analyzed 11-13 weeks postreconstitution, we found that the engraftment of the MLL-ELL-transduced cells was superior to that of the MIE controls. At this time point, the contribution of the donor cells was normally distributed among the myeloid and nonmyeloid compartments. Although all of the MIE animals (n = 10) remained healthy for more than a year, all of the MLL-ELL mice (n = 20) succumbed to monoclonal or pauciclonal acute myeloid leukemias within 100-200 days. The leukemic cells were readily transplantable to secondary recipients and could be established as immortalized cell lines in liquid cultures. These studies demonstrate the enhancing effect of MLL-ELL on the proliferative potential of myeloid progenitors as well as its causal role in the genesis of acute myeloid leukemias.

Molecular analysis of the T-cell acute lymphoblastic leukemia-associated t(1;7)(p34;q34) that fuses LCK and TCRB.

Previously we had characterized the t(1;7)(p34;q34) translocation from HSB-2. This translocation fused the beta T-cell receptor gene (TCRB) constant region and transcriptional enhancer with the type I transcription unit of the LCK gene on the derivative 1 [der(1)] chromosome. The type II promoter was translocated to the der(7) chromosome. Regarding the mechanism of the t(1;7) in HSB-2, we identified an alternating purine-pyrimidine tract (G-T)17 at the 1p34/LCK breakpoint. Additionally, sequence analysis of both breakpoint junctions provided data that implicate the V(D)J recombinase in formation of the t(1;7). A heptamer-nonamer recognition sequence with a 12-bp spacer was found in the immediate vicinity of the 1p34/LCK breakpoint and, thus, chromosomal breakage at 1p34 may be explained as resulting from recombinase activity. Because phosphorylation of Tyr-505 in vivo regulates the tyrosine kinase activity of p56lck we amplified a region from LCK exon 12 that contains the codon for Tyr-505 and showed no mutation of this codon in HSB-2 DNA and, therefore, p56lck in HSB-2 is not activated by mutation of Tyr-505. We have analyzed LCK gene expression in HSB-2 and SUP-T12 cell lines. RNase protection analysis identified almost exclusively type I transcripts in HSB-2. An independent t(1;7) in SUP-T12 also resulted in the juxtaposition of LCK to TCRB. The breakpoint in SUP-T12 occurred 2 kb 5' of the type II promoter, leaving an intact LCK gene on the der(1) chromosome. RNase protection analysis identified both type I and type II LCK transcripts in a 3:1 ratio in SUP-T12. Factors other than proximity to the TCRB enhancer must affect promoter utilization in this cell line.

Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia.

To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient's leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On "zoo blots," this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis.

DNA rearrangements and altered transcripts of the MLL gene in a human T-ALL cell line Karpas 45 with a t(X;11) (q13;q23) translocation.

Translocations involving chromosome band 11q23 are found in both lymphoid and myeloid leukemias as well as in lymphomas, in these translocations. The chromosomes most frequently involved in reciprocal translocations include chromosomes 4, 6, 9, and 19, and we and others have reported that chromosomes 1, 2, 10, 15, 17, 18, 22, and X are also involved. In the cell line Karpas 45, which has a t(X;11) (q13;q23) translocation, we report here that the MLL gene is rearranged and that there are two altered transcripts of MLL that come from the der(11) chromosome.

Abnormalities of chromosome band 11q23 and the MLL gene in pediatric myelomonocytic and monoblastic leukemias. Identification of the t(9;11) as an indicator of long survival.

PURPOSE AND METHODS: We reviewed the cytogenetic pattern of the malignant cells in 36 patients who were < 20 years of age and who had M4 and M5 leukemias, excluding M4Eo cases with inv(16). We performed fluorescence in situ hybridization (FISH) and molecular studies to determine the actual incidence of 11q23/MLL abnormalities in these patients. RESULTS: Eighteen patients had 11q23 translocations or insertions detected by cytogenetic analysis (15 cases) or by FISH (3 cases); 10 patients had t(9;11), all of whom had M5a. Eight patients had other 11q23 translocations or insertions not involving chromosome 9[t(11q23)] (four each had M4 or M5 leukemias). Eighteen cases with M4/M5 did not have 11q23 abnormalities. MLL rearrangements were found in all patients with translocations or insertions of 11q23 who were studied. Clinically, children with t(9;11) were indistinguishable from other patients with M4-M5 leukemias. In contrast, the t(11q23) group was characterized by extreme hyperleukocytosis, CNS disease, and skin involvement. Patients with the t(9;11) had a better outcome when compared with patients in the t(11q23) group (EFS +/- SE at 3 years, 56 +/- 17% versus 10 +/- 10%, p = 0.04), and to all the remaining children with M4-M5 leukemias (p = 0.04). CONCLUSIONS: The combination of cytogenetic, FISH, and molecular analysis provides a highly sensitive strategy for detection of 11q23/MLL gene rearrangements in childhood M4-M5 leukemias. Our more precise classification of these patients allows a more accurate correlation with outcome. The favorable prognostic significance of the t(9;11) should be confirmed in prospective studies including a larger number of children as well as adults.

Developmental analysis and subcellular localization of the murine homologue of ELL.

The ELL gene was first identified by its involvement with MLL in the translocation (11;19)(q23;p13.1) in acute myeloid leukemia. To date, nine other MLL partner genes have been cloned, but their precise functions have yet to be determined. To characterize the functions of ELL further, we have cloned the murine homologue of ELL and have found that the gene is highly conserved at the nucleotide and amino acid level. The open reading frame of the murine homologue contains 602 aa, slightly smaller than the 621 aa in the human gene. With Northern blot analysis, a 3.4-kb transcript is detected in all tissues examined with greatest levels of expression in the liver. Unlike human ELL, only a single transcript can be detected with either murine coding sequence or 3' untranslated region probes. To examine the spatial and temporal pattern of expression in murine development, in situ hybridization studies were performed with sense and antisense riboprobes from the 3' untranslated region of murine Ell. Ell is expressed diffusely by embryonic day 7.5 (E7.5). In addition, high levels of expression can be detected in maternally derived decidual tissue. At E14.5, Ell is expressed diffusely throughout the embryo. However by E16.5, specific expression in the liver and gastrointestinal tract becomes prominent and remains so in both neonates and adults. To determine the subcellular localization of ELL, we developed a polyclonal antiserum to ELL that was used for immunofluorescence studies in COS-7, HeLa, NIH 3T3, and A7r5 cells. The ELL protein was localized to the nucleus but excluded from nucleoli in all cell lines examined. Recently, the gene product of ELL was found to function as an RNA polymerase II elongation factor, an activity that is consistent with our immunofluorescence data. Thus, these studies extend our understanding of the normal functions of ELL and provide additional insight into its aberrant function when fused to MLL in acute myeloid leukemia.

EAF1, a novel ELL-associated factor that is delocalized by expression of the MLL-ELL fusion protein.

The (11;19)(q23;p13.1) translocation in acute leukemia leads to the generation of a chimeric protein that fuses MLL to the transcriptional elongation factor ELL. A novel protein was isolated from a yeast 2-hybrid screen with ELL that was named EAF1 for ELL-associated factor 1. Using specific antibodies, the endogenous EAF1 and ELL proteins were coimmunoprecipitated from multiple cell lines. In addition, endogenous EAF1 also exhibited the capacity to interact with ELL2. Database comparisons with EAF1 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that exhibited homology with the transcriptional activation domains of several translocation partner proteins of MLL, including AF4, LAF4, and AF5q31. A similar transcriptional activation domain has been identified in this region of EAF1. By confocal microscopy, endogenous EAF1 and ELL colocalized in a distinct nuclear speckled pattern. Transfection of the MLL-ELL fusion gene delocalized EAF1 from its nuclear speckled distribution to a diffuse nucleoplasmic pattern. In leukemic cell lines derived from mice transplanted with MLL-ELL-transduced bone marrow, EAF1 speckles were not detected. Taken together, these data suggest that expression of the MLL-ELL fusion protein may have a dominant effect on the normal protein-protein interactions of ELL.

A t(11;12) 11q23 leukemic breakpoint that disrupts the MLL gene.

Translocations involving 11q23 have been shown to be a consistent finding in human hematopoietic malignancies and in some constitutional abnormalities. The identification of a gene, MLL (myeloid/lymphoid or mixed-lineage leukemia), that spans the breakpoints in four different recurrent 11q23 translocations was recently reported. We describe a rare (11;12)(q23;p13) translocation, observed in leukemic cells from a patient with acute lymphoblastic leukemia, which also disrupts this gene.