Rearrangements In Leukemia Research Articles

Higher involvement of subtelomere regions for chromosome rearrangements in leukemia and lymphoma and in irradiated leukemic cell line

Importance of subtelomeric chromosome rearrangements associated with idiopathic mental retardation and with methylation of gene expression in neoplastic cells has been shown. In order to observe incidence of deletions or translocations involving subtelomere region in leukemias and lymphomas, 41 patients were observed precisely by chromosome metaphase fluorescent in situ hybridization (FISH) using subtelomere probes and so on, specially focus on the end of long arm of chromosome 11 or short arm of chromosome 17. The abnormalities of subtelomere region on chromosome 11 were frequently observed in 7 of 17 patients (41.6%) with 11q22-q25 abnormalities, which were 3 of 9 patients with add(11)(q23-q25) and 3 of 7 patients with del(11)(q22-25), and 16 of 24 patients (66.0%) with 17p13 abnormalities, which were 2 of 6 with translocation between 17 and other chromosome, 6 of 10 with add(17)(p13) and all of 8 with del(17)(p13). Lymphoid disease had slightly higher abnormalities of subtelomere region than myeloid disease in patients with 11q22-25 or 17p13 abnormalities. Chemo- or radiotherapy treated patients had also subtelomeric chromosomal rearrangements. These results indicate that leukemia and lymphomas have many abnormalities at subtelomere region and possible association of chromosome instability, relating to pathogenesis of these diseases especially after therapy. Furthermore, FISH and chromosome analyses were performed on long-term cultured HL-60 leukemic cell line after irradiation using three different sources, and the results confirmed the higher induction of chromosome instability involving subtelomere region after α-rays irradiation. Keywords: Chromosome instability, subtelomere, telomere, radiation, α-rays, γ-rays, β-rays, leukemia cell line, FISH analysis

Whole Genome Sequence Analysis of 22 MLL Rearranged Infant Acute Lymphoblastic Leukemias Reveals Remarkably Few Somatic Mutations: A Report From the St Jude Children‘s Research Hospital - Washington University Pediatric Cancer Genome Project

Abstract 69Infant (< 1 year of age) acute lymphoblastic leukemia (ALL) is a rare disease characterized by rearrangements of the Mixed Lineage Leukemia (MLL) gene at 11q23 and a poor prognosis. In an effort to determine the total complement of somatic mutations occurring in this high risk leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 22 infants with MLL rearranged ALL using the Illumina platform. In addition, we sequenced 2 paired relapse samples. Somatic alterations, including single nucleotide variations (SNV), and structural variations (SV) including insertions, deletions, inversion, and inter- and intra-chromosomal rearrangements were detected using complementary analysis pipelines including Bambino, CREST and CONSERTING. Validation of identified somatic mutations was performed using PCR amplification of the leukemia and germ line DNA followed by Sanger or 454-based sequencing, or by array-based capture followed by Illumina-based sequencing.Analysis of the structure of MLL rearrangements at the base pair level revealed that over half had complex rearrangements that involved either three or more chromosomes, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In five of the complex cases, chromosomal rearrangements were predicted to generate not only a MLL-partner gene fusion, but also novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B; MLLT10-CTNNAP3B; MLLT10-ATP5L / ATP5L-YPEL4; and CRTAM-GNL3. An analysis of the sequence surrounding the breakpoints of MLL and its partner genes suggest that the predominant mechanism of rearrangement involved non-homologous end joining.An analysis of the total number of non-silent mutations revealed infant ALL to have the lowest frequency of non-silent somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLL rearrangements, a mean of only 2 somatic SVs and 2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case, with a range of non-silent mutation of between 0 and 11 per case (0–7 SV and 0–5 SNV). Despite the paucity of mutations several pathways were recurrently targeted. Mutations leading to activation of signaling through the PI3K/RAS pathway was observed in 45% of the cases with mutation of individual components including KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and the GTPase activating protein ARHGAP32 (p200Rho/GAP), which mediates cross-talk between RAS and Rho signaling. Other pathways altered include B cell differentiation, with 23% of cases containing mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2.WGS of two infant ALL relapse samples and comparison with the data from their matched diagnostic samples revealed a marked increase in the number of mutations at relapse with additional SVs, SNVs, and CNAs identified. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone.Because of the exceedingly low frequency of mutations detected in infant ALL, we decided to define the frequency of non-silent SNVs in MLL rearranged leukemia occurring in older children (7–19 years of age). Exome sequencing was performed on 13 MLL leukemias (8 ALLs and 5 AMLs). This analysis revealed that non-infant pediatric MLL rearranged leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 8/case in older patients versus 2/case in infants, p<0.001). Although the increased frequency of mutations may be a reflection of the older age, the low number of cooperating mutations in infants raises the possibility that the target cell of transformation differs between infants and older children, with the cells present during early development requiring fewer cooperating mutations to induce leukemia.In summary our analysis demonstrated an exceedingly small number of mutations required to generate infant MLL rearranged leukemia. The number of detected somatic mutations may represent the lower limit of mutations required to transform a normal human cell into cancer. Disclosures:Fioretos:Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Chromosomal Minimal Critical Regions in Therapy-Related Leukemia Appear Different from Those of De Novo Leukemia by High-Resolution aCGH

Therapy-related acute leukemia (t-AML), is a severe complication of cytotoxic therapy used for primary cancer treatment. The outcome of these patients is poor, compared to people who develop de novo acute leukemia (p-AML). Cytogenetic abnormalities in t-AML are similar to those found in p-AML but present more frequent unfavorable karyotypes depending on the inducting agent. Losses of chromosome 5 or 7 are observed after alkylating agents while balanced translocations are found after topoisomerase II inhibitors. This study compared t-AML to p-AML using high resolution array CGH in order to find copy number abnormalities (CNA) at a higher resolution than conventional cytogenetics. More CNAs were observed in 30 t-AML than in 36 p-AML: 104 CNAs were observed with 63 losses and 41 gains (mean number 3.46 per case) in t-AML, while in p-AML, 69 CNAs were observed with 32 losses and 37 gains (mean number of 1.9 per case). In primary leukemia with a previously “normal” karyotype, 18% exhibited a previously undetected CNA, whereas in the (few) t-AML with a normal karyotype, the rate was 50%. Several minimal critical regions (MCRs) were found in t-AML and p-AML. No common MCRs were found in the two groups. In t-AML a 40kb deleted MCR pointed to RUNX1 on 21q22, a gene coding for a transcription factor implicated in frequent rearrangements in leukemia and in familial thrombocytopenia. In de novo AML, a 1Mb MCR harboring ERG and ETS2 was observed from patients with complex aCGH profiles. High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML; those more frequently found in t-AML and those specifically found in p-AML.

Cytotoxicity, drug combinability, and biological correlates of ABT‐737 against acute lymphoblastic leukemia cells with MLL rearrangement

ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 to induce apoptosis in malignant cells and has shown promise as an effective anti-leukemic agent in pediatric preclinical tests. This study focuses on the effects of ABT-737 on leukemia cells with MLL rearrangement and identifies some of the biological correlates of its activity. Cells were cultured in the presence of increasing concentrations of ABT-737 alone or in combination with other agents. After 4 days in culture, cell growth inhibition was measured by Alamar blue assay. The expression and activation of potential intracellular targets of ABT-737 activity were determined by Western blot analysis. Significant Bcl-2 expression was detected in all infant leukemia cells investigated. ABT-737 induced cell death in all cell lines studied although the IC(50) values differed somewhat between cell lines. Western blot analysis identified the effects of ABT-737 on survival and apoptosis-regulatory proteins PARP, caspase-8, and cytochrome-c. Drug combination studies indicated synergy with distinct anti-neoplastic agents, including the multi-tyrosine kinase inhibitor sunitinib. This effective drug synergy appears to be mediated by the combined inhibition of Bcl-2 and intracellular signaling pathways. We describe the in vitro studies to demonstrate the activity and drug combinability of ABT-737 against MLL rearranged leukemia cells. In addition, identification of the molecular changes that occur in the presence of ABT-737 provides information regarding effective target validation and target modulation analyses in future clinical trials.

NPM1 Deletion Is Associated with Gross Chromosomal Rearrangements in Leukemia

Background NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated.Methodology and Principal FindingsBone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the “5q- syndrome” but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p = 0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion.Conclusions and Significance NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.

Abstract 3886: Therapeutic potential of CDK inhibitors in MLL leukemias

Abstract Chromosomal rearrangements involving the human mixed - lineage leukaemia (MLL) gene at 11q23, are associated with the development of acute leukemias. Abnormalities in the MLL gene can be detected in de novo acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) as well as in therapy-related AML, particularly after treatment with DNA topoisomerase II inhibitors. Some malignancies, such as infant ALL, are characterised by exceedingly high incidence (∼80%) of MLL gene rearrangements, which is a strong predictor of an adverse outcome. Current therapies fail in &amp;gt;50% of the infant MLL cases, indicating that innovative therapeutic strategies are urgently needed to improve prognosis. Re-arranged MLL interacts with the transcription complex including CDK9. The association of CDK9 results in an increase in RNApol II phosphorylation and hence overexpression of HoxA9 and Meis1, which are normally down regulated during haematopoietic differentiation, resulting in immortalisation and transformation. These suggest a key role of CDK9 activity in this transformation process and the potential of CDK9 inhibitors to stop the uncontrolled myeloblast proliferation. Additionally, the expression of MCL-1, an anti-apoptotic member of BCL-2 family, was shown to increase in infant MLL samples, which correlated with prednisone resistance and poor prognosis. Importantly, MCL-1 also plays an important role in the development of B and T lymphocytes as well as in the survival of haematopoietic stem cell. Therefore, targeting MCL-1 in infant MLL cells may also induce leukemic cell death of the CD34+CD19- chemoresistant leukemic stem cell. The ability of CDK inhibitors, such as seliciclib, flavopiridol, SNS-032, to down regulate MCL-1 and induce apoptosis in B-CLL and MM cells is well recorded. We studied the effect of a purine CDK inhibitor, Cmpd 5, on MLL cell lines in vitro and in vivo. Cmpd 5 is a structural analogue of seliciclib and a potent and selective inhibitor of CDKs 2, 5, and 9. AML cell lines with MLL re-arrangements were exquisitely sensitive to Cmpd 5. Treatment for 5 hours at concentrations &amp;lt;0.5 μM was sufficient to inhibit completely the proliferation of the most sensitive cell lines. The mode of action corresponded to the target inhibition and included rapid induction of apoptosis. Higher concentration and longer treatment were required to achieve similar effect in a WT MLL cell line. The high sensitivity of MLL/AML cell lines was confirmed in vivo. A very potent (97% TGI) and durable effect was observed in a mouse xenograft model after oral administration of Cmpd 5. Our results suggest that MLL rearranged leukemias are very sensitive toward CDK inhibitors, which down regulate transcription, and justifies further exploration of their therapeutic potential for MLL haematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3886.

FLT3, NPM1 and MLL Mutations Help Risk Stratification in Pediatric Acute Myeloid Leukemia.

Abstract 1574▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-600 IntroductionAcute myeloid leukemia (AML) is a heterogeneous disease which harbors various genetic alterations. Among theses genetic events, Mutations of FLT3, NPM1, MLL and other genes often predict prognosis, particularly in cases cytogenetic normal (CN-AML). Could these be criteria for risk stratification in Pediatric AML ? Patients and Methods155 cases of de novo AML were diagnosed routinely according to morphology, immunology, cytogenetics, and molecular biology examination on bone marrow (BM) aspirates between Jan. 2002 and Dec. 2008. All patients received chemotherapy according to the AML-XH-99 protocol, which consist of Daunorubicin, Cytosine arabinoside, Etoposide, Homoharringtonine. For acute promyelocytic leukemia, all-trans retinoic acid and Arsenic trioxide were also included. Meanwhile, total RNA of leukemic cells form all diagnostic BM samples were extracted, and then reverse transcribed. MLL partial tandem duplication (MLL/PTD) fusion transcripts were screened by real-time quantitative polymerase chain reaction. FLT3 internal tandem duplication (FLT3/ITD), FLT3 tyrosine kinase domain mutation (FLT3/TKD) and NPM1 mutation were examined by High resolution melting analysis. ResultsOf the 155 children with de novo AML, 121(78.1%) had received chemotherapy for more than one week with data available for analysis. Among them, 55(45.5%) was cytogenetically normal (CN-AML). In this total cohort of patients 49(27.09%) had FLT3/ITD (32.70% in CN-AML), 14 (9.03%) had FLT3/TKD (7.30% in CN-AML), 62 (40%) had NPM1 mutation (49% in CN-AML), and additional 8 (5.16%) had MLL/PTD (5.50% in CN-AML). In this cohort of patients 98 (63.22%) had at least one mutation. The clinical outcomes were listed in table 1. Generally, patients with FLT3 mutation (ITD or TKD mutation) usually have worse results after chemotherapy, as reported previously by other researchers. Meanwhile, NPM1 mutations usually predict better prognosis in our cohort of AML patients. MLL/PTD always predicts the worst outcome in AML as other MLL rearrangements in leukemia. Among CN-AML patients, 5-year EFS and OS were similar to whole cohort of patients according to those mutations. Cox regression analysis in a univariate model revealed that the presence of FLT3/ITD and NPM1 was significant prognostic factor of EFS, (P<0.05). We therefore proposed a molecular-risk classification of pediatric AML patients based on the data we got in this study. For the newly classified groups of low, medium and high risk groups, EFS rate was 62.03%±8.42%, 45.42%±4.52%, and 14.85%±2.99%, respectively, P=0.00. CRD for the 3 groups was 27.69±21.34 months, 22.62±19.64 months, 13.26±11.95 months, respectively, p=.022. Our results indicate that combinations of these couple of molecular events may be the useful tool for further classify AML in children.Table 1The clinical outcomes of pediatric AML patients with different gene mutationsMutations/wildFirst course CR (%)5-year EFS (%)5-year OS (%)CR Duration (Month)FLT3/ITD50/76.5 (p = 0.004)24.65 ± 3.93/55.01 ± 5.09 (p = 0.007)49.13 ± 7.08/62.24 ± 4.96 (p = 0.173)18.58 ± 17.37/23.71 ± 20.09 (p = 0.185)FLT3/TKD72.7/68.2 (p = 0.527)43.68 ± 9.79/50.48 ± 4.319 (p = 0.373)49.21 ± 9.25/60.595 ± 4.31 (p = 0.233)38.45 ± 29.84/20.55 ± 17.05 (p = 0.003)NPM1 Mutation72.3/66.2 (p = 0.308)61.41 ± 6.26/36.30 ± 4.24 (p = 0.014)75.45 ± 5.29/47.12 ± 5.18 (p = 0.001)27.15 ± 20.93/19.03 ± 17.78 (p = 0.024)MLL/PTD25/70.1 (p = 0.091)8.50 ± 3.10/50.98 ± 4.18 (p = 0.04)14.33 ± 1.76/60.39 ± 4.19 (p = 0.091)8.5 ± 6.19/55.65 ± 19.53 (p = 0.024) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MLL Fusion Proteins Directly Regulate a Small Set of Wild Type MLL Target Genes.

Abstract 1279Poster Board I-301The MLL (mixed-lineage leukemia) gene at chromosome band 11q23 is rearranged frequently in AML and ALL, and associated with poor prognosis. The consequence of these translocations is the formation of a chimeric oncogenic transcription factor that specifies a unique expression signature distinct from other subtypes of acute leukemia. However, it is poorly understood, which changes in gene expression in leukemic cells are under the direct control of MLL fusion proteins (fusion), nor is it clear what is the potential overlap between MLL wild type (WT) and fusion target genes. In the present study, we used genome-wide location analysis to determine the genomic loci that are specifically bound by MLL fusion proteins. Combining the binding analysis with expression profiling, we further defined the subset of MLL fusion-bound genes whose expression is regulated by the presence of MLL fusion proteins. Using ChIP-chip (Chromatin Immunoprecipitation coupled with micro-array), we determined the MLL-bound regions in 5 myeloid leukemic cell lines using a custom array containing the entire genomic region of 200 genes previously found to have altered expression in MLL-rearranged leukemias. Examination of these 200 genomic loci revealed a largely overlapping set of genes bound by MLL (wild type and/or fusion proteins) in WT/WT (U937: 110 genes, HL60: 79 genes) and WT/Fusion cells (MV4;11: 62 genes, THP-1: 89 genes). Surprisingly, the MLL-bound genes in fusion/fusion (ML-2) cells (25 genes) are a small subset of that found in each of the other 4 cell lines, despite comparable levels of detected MLL binding signal across all lines examined. These data suggest that the MLL fusion protein is likely only localized to a limited portion of genomic loci occupied by the MLL wild type protein. To test this hypothesis in a more systematic way, we examined an inducible MLL-ENL–ER transformed cell line (Slany et al, MCB 2004), which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. As MLL-ENL promotes histone H3 lysine 79 (H3K79) methylation, we determined both MLL binding and H3K79 methylation using a genome-wide location analysis. We anticipated that MLL-fusion bound genomic regions would exhibit a significant drop in either MLL and/or K79 signal upon inactivation of MLL-ENL. Unexpectedly, among thousands of genes that are bound by MLL, only 10% of them (222 genes) showed a pattern of binding increase between MLL-ENL induced and un-induced conditions. To explore the impact of MLL fusion protein on gene activation, we performed whole genome expression profiling in the presence or absence of MLL-ENL. Increased levels of either MLL binding or H3K79 methylation are significantly associated with differential gene expression. Among 222 MLL fusion target genes, 12 of them are differentially up-regulated in the presence of MLL-ENL, indicating that a large fraction of MLL fusion bound genes do not exhibit significant changes in mRNA expression. The identified 12 genes include key regulators in cellular differentiation and cell cycle regulation, as well as Meis1, Hoxa9 which are known to be essential for the development of MLL leukemia. To explore the apparent discrepancy between the massive expression changes in MLL rearranged leukemia and the small number of direct fusion target genes we identified, we tested the hypothesis that a significant portion of the MLL fusion protein expression signature was derived from its direct fusion target genes Meis1 and Hoxa9. Using publicly available data, we compared the MLL leukemia associated expression profile with the set of genes that were down-regulated upon knock-down of Meis1 and Hoxa9. We found significant enrichment of Hoxa9/Meis1 downstream targets in the expression profile defined by MLL fusion proteins. Altogether, our data suggest that MLL fusion proteins are likely to contribute to the development of acute leukemia through direct activation of a very small set of genes. The results have important implications in understanding the mechanisms of target gene specificity involving oncogenic transcription factors. DisclosuresNo relevant conflicts of interest to declare.

Consistent Deregulation of Gene Expression between Human and Murine MLL Rearrangement Leukemias

Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the down-regulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis.

Controversies of and Unique Issues in Hematopoietic Cell Transplantation for Infant Leukemia

Infants with leukemia who require hematopoietic cell transplantation (HCT) remain 1 of the most significant challenges in pediatric stem cell transplant. Infant leukemia is characterized by a unique biology including a predominance mixed lineage leukemia(MLL) gene rearrangement and juvenile myelomonocytic leukemia. Moreover, the long-term effects of transplantation are particularly prominent in infants who have active endocrine, cardiovascular, pulmonary, neurologic, musculoskeletal, hearing, and vision development, with the added risk of second malignant neoplasms. Currently, there is no solid basis to support allogeneic HCT as first-line therapy for infant acute lymphoblastic leukemia-first remission (ALL-CR1), although indicated for other infant leukemias, including juvenile myelomonocytic leukemia (JMML). The relative long-term toxicity of total body irridiation (TBI) versus non-TBI containing preparative regimens for HCT in infants remains controversial, with the differences, especially on neurocognitive function, unknown.

A new fusion gene NUP98-IQCG identified in an acute T-lymphoid/myeloid leukemia with a t(3;11)(q29q13;p15)del(3)(q29) translocation

NUP98 has been involved in multiple recurrent chromosome rearrangements in leukemia. We identified a novel fusion between NUP98 and IQ motif containing G (IQCG) gene from a de novo acute T-lymphoid/myeloid leukemia harboring t(3;11)(q29q13;p15)del(3)(q29). IQCG has two putative coiled-coil domains and one IQ domain. The FG repeat from NUP98 and the coiled-coil domain from IQCG were retained in the fusion protein. We demonstrated that NUP98-IQCG could form homodimer, heterodimerize with NUP98 or IQCG, bind co-activators and/or co-repressors, and show transcriptional activity in vitro. Expression of NUP98-IQCG inhibited 32Dcl3 cell apoptosis induced by Ara-C, and partially blocked granulocyte differentiation induced by G-CSF. Colony-forming assay and serial replating assays indicated that NUP98-IQCG was able to stimulate proliferation, partially block differentiation of hematopoietic stem/progenitor cells but was unable to confer transformation alone. Taken together, our data indicate that newly identified NUP98-IQCG fusion protein may play an essential role in leukemogenesis, but by itself may not be sufficient to induce leukemia.

A New Fusion Gene NUP98-IQCG Identified in an Acute T/Myeloid Leukemia with t(3;11)(q29q13;p15) Translocation.

NUP98 has been involved in multiple recurrent chromosome rearrangements in leukemia. We identified a novel fusion between NUP98 and IQ motif containing G (IQCG) from a de novo acute T/myeloid leukemia harboring t(3;11)(q29q13;p15). IQCG has 2 putative coiled-coil domains and an IQ domain. The FG repeat from NUP98 and the coiled-coil domain from IQCG were retained in the fusion protein. We demonstrated that NUP98-IQCG could form homodimer or heterodimerize with either NUP98 or IQCG, bind Co-activators and/or Co-repressors, and show transcriptional activity in vitro. Expression of NUP98-IQCG inhibited 32Dcl3 cell apoptosis induced by Ara-C, and partially blocked granulocyte differentiation induced by G-CSF. Colony forming assay also indicated that NUP98-IQCG was able to stimulate proliferation and partially block differentiation of hematopoietic stem/progenitor cells. We conclude that NUP98-IQCG may play an essential role in leukemogenesis by stimulating progenitor cell proliferation and inhibiting differentiation and apoptosis.

A Novel Approach for Analyzing Gene Expression Profiles Defines Two Distinct Subgroups of t(4;11) Positive Infant Acute Lymphoblastic Leukemia Patients.

Gene expression profiles (GEPs) are becoming increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome for various types of cancer. However, established gene signatures not always appear helpful in understanding underlying disease mechanisms. Therefore, we explored an alternative approach for analyzing GEPs of a group of t(4;11) positive infant acute lymphoblastic leukemia patients (n=15), a highly aggressive type of MLL rearranged leukemia. We developed a method that uses a relational database program in combination with a normalization approach and specific discriminators. For normalization, every GEP of a given t(4;11) positive ALL sample was compared with 3 GEPs of normal bone marrow aspirates derived from healthy donors. Using the GeneChip Analysis Suite 5.0 program for single comparison analysis, the resulting gene lists were then compared with each other and only consistently identified genes (present on all three gene lists) were used for further analysis. This noise reduction decreased the amount of potentially deregulated target genes to about 30–40%. The remaining gene lists represented highly significant target genes that were then incorporated into a relational database program using specific discriminators. These discriminators were:1.upregulation of HOXA9 and HOXA10,2.presence/absence of the AF4-MLL fusion transcripts in addition to the MLL-AF4 fusion, and3.the localization of the genomic breakpoint within the MLL gene.This pilot study led to promising results, surprisingly classifying individual t(4;11) positive ALL patients into two distinct subgroups. Both subgroups share about 80 target genes, but also display particular sets of subgroup specific target genes. Importantly, these identified target genes can directly be linked to biological properties of t(4;11) positive leukemia cells, and therefore, allow important novel insights into this aggressive type of leukemia in infants.

Genome-Wide Profiling of High-Risk Pediatric Acute Lymphoblastic Leukemia (ALL): The ALL Pilot Project for the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) Initiative.

Recent studies have identified a high frequency of recurrent acquired DNA copy number abnormalities in pediatric ALL, most commonly involving genes regulating lymphoid development (Nature 2007; 446:758). The majority of B-progenitor ALL cases in this study harbored recurring chromosomal abnormalities such as hyperdiploidy and recurring translocations, many of which are associated with favorable outcome. Data from large cohorts of poor risk B-ALL have not been reported. Here we report genomic profiling data from a cohort of 221 pediatric ALL cases treated on the Children's Oncology Group P9906 study from 2000–2003 with an augmented BFM regimen (N Engl J Med 1998; 338:1663). The study targeted poor prognosis ALL cases, using age/WBC criteria designed to identify a subset of patients with NCI high risk ALL that historically had a very poor outcome. Cases with favorable (trisomy 4 + 10; ETV6-RUNX1) or unfavorable (BCR-ABL1 or hypodiploid) genetic features were excluded. The cohort included 25 TCF3-PBX1 and 19 MLL-rearranged cases. Profiling of DNA copy number abnormalities was performed using Affymetrix 250k Sty and Nsp arrays, reference normalization, dChipSNP, and circular binary segmentation. Germline SNP array data was available for 210 cases. The most frequent abnormalities were deletions of CDKN2A in 101 cases (45.7%), the B-lineage transcription factors PAX5 (N=68, 30.5%) and IKZF1 (Ikaros, N=32, 14.5%), ETV6 (N=29, 13.1%), RB1 (N=25, 11.3%), BTG1 (N=23, 10.4%), the tumor suppressor candidate TSC22D1 (N=20, 9%), the microRNA cluster at 13q14 (N=19, 8.6%), and deletion (N=3) or amplification (N=20, 4 focal, 16 broad) of the retinoic acid pathway gene CCDC26. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, EBF, IL3RA, ERG, TOX, RAG1/2, KRAS, NRAS, NR3C2 and the adenosine deaminase pathway genes ADAR and ADARB2. An unexpected finding was focal deletion involving DMD (dystrophin) at Xp21.1 in 15 (6.8%) cases. Copy number abnormalities were uncommon in MLL rearranged leukemias, suggesting that fewer secondary mutations are needed for leukemogenesis in this genetic subtype of ALL. Clustering of copy number data using non-negative matrix factorization identified 11 clusters of cases, including clusters driven by isochromosome 7q, deletions of 9p, gains of 1q, multiple whole chromosomal gains, and intrachromosomal amplification of chromosome 21 (iamp21). These findings confirm the high frequency of deletions involving genes regulating B cell development and cell cycle such as PAX5 and CDKN2A in both standard and poor risk B-ALL. Furthermore ETV6 (TEL) deletions are shown to be common in B-ALL cases lacking the ETV6-RUNX1 translocation. The identification of novel recurring abnormalities (IL3RA, KRAS, NRAS) emphasizes the importance of high resolution copy number analysis in leukemia. These data are being integrated with gene expression data to select genes for resequencing as part of the TARGET project.

HOXA9 Represses Bim Expression in MLL Rearranged Leukemia: Implications for Drug Therapy.

MLL translocations can be found in roughly 10% of acute leukemias, including over 70% of infant leukemias. These leukemias are often associated with a poor prognosis, making new therapeutics for this subtype of leukemias a priority. HoxA cluster genes have been shown to be upregulated by MLL fusion proteins, and make up a portion of a self-renewal associated gene expression signature found in murine models of MLL-AF9-expressing leukemia stem cells. The role of HoxA cluster genes in MLL-fusion mediated leukemia is not clearly understood, mainly due to lack of knowledge of HoxA downstream targets. One possible role for HoxA proteins in MLL-fusion induced leukemia is enhancement of growth and survival pathways. Evidence for this possibility can be found in C.elegans, where the pro-apoptotic BH3-only gene egl-1 is regulated by homeobox genes. Therefore we sought to determine whether a HoxA cluster gene could in fact regulate survival/apoptosis machinery. Here we demonstrate that HoxA9 serves as a repressor of the pro-apoptotic BH3-only family member Bim. Knockdown of HoxA9 using shRNA in the MLL-AF9 expressing AML cell line Molm14 leads to upregulation of Bim, and rapid induction of apoptosis. Interestingly, knockdown of Meis1 or Pbx2, known binding partners of HoxA9, showed only an intermediate effect, with slower growth, but without an increase in Bim levels. Other BH3-only family members Noxa and Puma showed no significant change in expression after HoxA9 suppression as determined by quantitative RT-PCR. Using a FLAG-HOXA9 expressing Molm14 cell line, we were able to determine by chromatin immunoprecipitation that HOXA9 is present at a DNA region containing a canonical HOX-PBX binding site (TGATTTAT) roughly 100bp downstream of the first exon-intron junction in the human Bim gene. As histone deacetylases (HDACs) are generally transcription repressors, it is plausible that HOXA9 recruits HDACs to the Bim gene, thus regulating its expression. Since HDAC inhibitors also activate BIM, we assessed BIM activation in Molm14 cells after treatment with an HDAC inhibitor, and noted similar kinetics of BIM activation, suggesting a possible link between HoxA9 mediated suppression of BIM and HDACs. We are now assessing HOXA9/HDAC interactions in MLL rearranged leukemia cells. These data suggest that HOXA9 ensures survival of leukemia cells by directly inhibiting transcription of the pro-apoptotic gene Bim, and that interfering with this mechanism may improve treatment for MLL-fusion bearing leukemias.

Distinct Methylation Patterns among Infants with MLL Rearranged Acute Lymphoblastic Leukemia.

Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) is characterized by a high incidence of rearrangements of the MLL gene (∼80%) which is associated with a poor prognosis. The most frequent MLL rearrangements in infant ALL are translocations t(4;11), t(11;19) and t(9;11). Recently, gene expression profiling has established MLL rearranged leukemia as a unique type of leukemia (denoted MLL), that is clearly distinguishable from other ALL subtypes. Currently, these gene expression profiles are slowly revealing important genetic properties underlying this aggressive type of leukemia, however, any epigenetic data on MLL are still lacking. Therefore, the present study was designed to unravel the MLL-specific methylation patterns underlying infant MLL by applying differential methylation hybridization (DMH) using CpG island microarrays containing ∼9000 CpG island probes, in duplicate. Primary infant ALL samples carrying t(4;11) (n=21), t(11;19) (n=17) and t(9;11) (n=6) were compared to infant ALL (n=13) and non-infant pediatric ALL (n=15) samples without MLL rearrangements. In addition, healthy pediatric bone marrow samples (n=9) were included as a reference. Compared to healthy controls, 656 CpG island probes were identified as significantly hypermethylated in t(4;11) positive samples, and 131 CpG island probes in t(11;19) positive samples (p<0.01, false discovery rate <5%). Interestingly, t(11;19) positive ALL patients shared 95% of their methylated probes with t(4;11) patients, suggesting a common methylation pattern which is completely absent in both infant and non-infant ALL patients lacking MLL rearrangements. Remarkably, displaying only a single probe significantly methylated as compared to healthy bone marrow, this common methylation pattern is also absent in t(9;11) positive ALL patients, indicating that based on genome-wide methylation, these patients represent a distinct entity clearly distinguishable from other MLL subgroups. Moreover, the fact that t(4;11) patients exhibit 532 methylated CpG island probes that were not found to be methylated in t(11;19) patients, demonstrates that these patients also exhibit a t(4;11) specific set of methylated genes. Identification of the genes represented by these CpG island probes and subsequent validation of the results obtained in this study is currently being performed (using pyrosequencing and methylation specific PCR analyses). In conclusion, these data reveal that different types of MLL rearranged infant ALL show distinct genome-wide methylation patterns. Specifically, infant ALL patients carrying t(4;11) and t(11;19) are characterized by severe CpG island hypermethylation, as compared to both t(9;11) positive infant ALL patients, as well as pediatric ALL patients lacking MLL rearrangements. Therefore, t(4;11) and t(11;19) patients in particular may well be suitable candidates for DNA methylation inhibiting therapeutic intervention. Finally, these promising results for the first time provide epigenetic insights into the complex biology of infant MLL, and clearly warrant further investigation currently being performed at our laboratory.

Down-regulation of DLX3 expression in MLL-AF4 childhood lymphoblastic leukemias is mediated by promoter region hypermethylation

Hypermethylation of CpG islands is the most well defined epigenetic change in neoplasia and plays an important role in the inactivation or silencing of cancer related genes. DLX genes (1-7), with large CpG islands in their 5' region, are implicated in a number of processes among which haematopoiesis. They are characterized by highly dynamic spatio-temporal expression and supposed to be involved in resistance to apoptosis of several tumor cell lines. In acute lymphoblastic leukemia (ALL) hypermethylation is a common phenomenon frequently associated with poor prognosis in specific genetic childhood leukemia subgroups. These data together with the presence of large CpG islands in the up-stream regions of the DLX genes make them attractive candidates for methylation regulated gene expression and leukemia related aberrancies. To validate the role of DLX genes in paediatric B-ALL cells, we studied two cell lines and two groups of patients with paediatric chromosomal rearrangements: MLL-AF4 and TEL-AML1, respectively. Analysis of methylation and gene expression patterns of DLX3 in 64 specimens of B-lineage ALL revealed that DLX3 presents aberrant methylation in paediatric B-ALL patients. In vitro experiments with 5-Aza-2'dC on leukemia cell lines, confirmed by Western blot analysis, indicated that the methylation of DLX3 CpG islands has a functional role and interferes with the DLX3 gene and DLX3 protein expression in B-ALL cells. Importantly, hypermethylation of DLX3 significantly reduces its expression in MLL-AF4 rearranged leukemias while methylation is almost absent in TEL-AML1 positive ALL specimens. These results show that differential DLX3 methylation could be a new epigenetic marker for genotypic B-cell leukemia subgroup with high-risk features.

Epigenetic regulation of brain expressed X-linked-2, a marker for acute myeloid leukemia with mixed lineage leukemia rearrangements

Epigenetic regulation of brain expressed X-linked- 2, a marker for acute myeloid leukemia with mixed lineage leukemia rearrangements

HoxA9 Knockdown Inhibits Proliferation and Induces Cell Death in Human MLL-Rearranged Leukemias.

Homeobox containing (Hox) genes are implicated in the regulation of normal and leukemic hematopoesis. Using gene expression profiling we and others have previously shown that HoxA9 is highly expressed in lymphoid and myeloid leukemias harboring MLL translocations and that high level HoxA9 expression is associated with poor clinical prognosis. Furthermore, HoxA9 plays variable roles in MLL-fusion induced murine leukemias. In this study we aimed to elucidate the role of aberrant HoxA9 expression in human MLL-rearranged and non-rearranged leukemia's utilizing an shRNA mediated knockdown approach. To establish an efficient knockdown assay three different shRNA constructs targeting human HoxA9 were synthesized and stably introduced into t(9;11) MOLM14 cells utilizing a lentiviral vector system. The shRNA construct which showed highest efficiency as measured by Taqman quantitative PCR (75–80% knockdown MLL-AF9 RNA) and Western Blot analysis was used for further experiments. In MOLM-14 cells, HoxA9 directed shRNA inhibited cell proliferation starting as early as 48h after transduction as determined by MTT assay, and at 72h demonstrated a markedly increased number of apoptotic cells as measured by Annexin V staining. This effect was rescued by introducing a non-targetable exogenous HoxA9 in MOLM-14 cells. To investigate if the HoxA9 knockdown related effects are specific for MLL rearranged cells we next analyzed cell growth and viability in 17 AML/ALL cell lines (7 MLL-rearranged, 10 non rearranged) after shRNA mediated HoxA9 knockdown. Interestingly, impaired cell proliferation and induction of apoptosis was significantly higher in the MLL rearranged cell lines (mean viability: 51.88%) than in the non-rearranged cells (mean viability: 90.98%; p=0.007). Moreover, the effect was also significantly correlated with the baseline HoxA9 mRNA expression before knockdown, with the greatest effect in cell lines expressing the highest HoxA9 levels (R= 0.8, p=0.00017). These findings prompted us to further analyze the effect of HoxA9 knockdown in MLL rearranged and non-rearranged primary human AML cells (6 MLL rearranged, 6 MLL germline). Similar to our findings in cell lines, we found a significantly higher effect on cell proliferation/viability in association with the presence of an MLL translocation (p=0.005) and a significant correlation with the baseline HoxA9 mRNA expression (R= 0.8, p=0.001). Next, we assessed the in vivo effect of HoxA9 knock down by transplanting luciferase-expressing SEMK2 (t4;11) cells and subsequent bioluminescent imaging. SEMK2 cells were transduced with either HoxA9 directed or control shRNA and intravenously injected into SCID-beige mice.Reconstitution was confirmed by in vivo bioluminescent imaging. 34 days after transplantation all mice in the HoxA9 shRNA group (n=4) are still alive with no signs of leukemia whereas all mice in the control group (n=3) have succumbed. Taken together our data implicates an important role for aberrant HoxA9 expression in human MLL rearranged leukemia cells.

Induction of Bim Facilitates Apoptosis in Leukemia Cells Treated with HDAC Inhibitors.

Lymphoblastic leukemias containing chromosomal translocations involving the Mixed Lineage Leukemia (MLL, HRX, ALL-1) gene, as well as most acute myeloid leukemias (AMLs) have relatively poor clinical prognoses due in part to intrinsic drug resistance. Therefore, new avenues are being explored for treatment of MLL-rearranged ALL and AMLs. One possible new therapeutic class currently being investigated is the histone deacetylase (HDAC) inhibitors. We utilized the histone deacetylase inhibitor NVP-LAQ824 (Novartis, Basel, Switzerland) and analyzed its effects on MLL rearranged and other myeloid leukemias. We also made use of an MLL-AF9 expressing myeloid leukemia cell line (AKLG) derived from purified murine leukemia stem cells to perform gene expression analysis on NVP-LAQ824 treated cells in order to further understand the mechanism of action of HDAC inhibitors, and to potentially identify cooperating therapeutics. NVP-LAQ824 inhibits cell growth at similar concentrations for all cell lines and primary patient samples tested (~25–50nM) as determined by MTT assay 48 hours after treatment. NVP-LAQ824 does not appear to induce apoptosis solely through inhibition of the HSP90/FLT3-ITD complex as cell lines possessing FLT3-ITD (a HSP90-chaperoned protein) and cells without this mutation have similar drug sensitivities. In fact, in cells overexpressing FLT3-ITD that are treated with NVP-LAQ824, phospho-FLT3-ITD levels do not diminish. Microarray data indicated that NVP-LAQ824 induces the BH3-only family member bim. This finding was verified by Western blotting in all cell lines and patient samples tested. Further, shRNA-mediated knockdown of Bim induced relative resistance of cells to NVP-LAQ824. The expression profile also showed similarities to gene expression patterns of dexamethasone treated cells, namely, increased bim levels and decreased expression of c-myc, raising the possibility of synergy between the two drugs. Using MTT assays, we discovered that NVP-LAQ824 in low doses (25nM) induces sensitivity to dexamethasone in glucocorticoid resistant cell lines in a glucocorticoid receptor (GR) dependent manner. Therefore, our data indicate that NVP-LAQ824 may reverse glucocorticoid resistance and may provide insight into glucocorticoid resistance in MLL rearranged leukemias. The biochemistry behind HDAC inhibitors merits further study.